Prion protein ligands and methods of use

ABSTRACT

Ligands that bind to prion proteins and methods for using the ligands for detecting or removing a prion protein from a sample, such as a biological fluid or an environmental sample. The ligands are capable of binding to one or more forms of prion protein including cellular prion protein (PrPc), infectious prion protein (PrPsc), and recombinant prion protein (PrPr). Prions from various species, including humans and hamsters, are bound by the ligands. Also provided is a method of treating or retarding the development of a prion-associated pathology in a subject

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional PatentApplication Ser. No. 60/430,423 filed Dec. 3, 2002.

FIELD OF THE INVENTION

[0002] This invention relates to the field of protein-ligandinteractions and more particularly relates to the identification ofligands that bind to prion proteins and methods of using the ligands todetect or remove prions from biological samples.

BACKGROUND OF THE INVENTION

[0003] Native or cellular prion protein “PrPc” is widely distributedthroughout the mammalia and has a particularly well-conserved amino acidsequence and protein structure. Infectious prions are thought to becomposed of a modified form of the normal cellular (PrPc) prion proteinand are called “PrPsc”. Prions have some properties in common with otherinfectious pathogens, but do not appear to contain nucleic acid.Instead, it is proposed that a post-translational conformational changeis involved in the conversion of non-infectious PrPc into infectiousPrPsc during which α-helices are transformed into β-sheets. PrPccontains three α-helices and has little β-sheet structure; in contrast,PrPsc is rich in β-sheet. The conversion of PrPc to PrPsc is believed tolead to the development of transmissible spongiform encephalopathies(TSEs) during which PrPsc accumulates in the central nervous system(CNS) and is accompanied by neuropathologic changes and neurologicaldysfunction. PrPsc, often referred to as the “scrapie” form of the prionprotein, is considered necessary and possibly sufficient for thetransmission and pathogenesis of these transmissible neurodegenerativediseases of animals and humans.

[0004] Specific examples of TSEs include scrapie, which affects sheepand goats; bovine spongiform encephalopathy (BSE), which affects cattle;transmissible mink encephalopathy, feline spongiform encephalopathy andchronic wasting disease (CWD) of mule deer, white-tailed deer,black-tailed deer and elk. In humans TSE diseases may present themselvesas, kuru, Creutzfeldt-Jakob disease (CJD),Gerstmann-Straüssler-Scheinker Syndrome (GSS), fatal insomnia andvariant Creutzfeldt-Jakob disease (vCJD). vCJD recently emerged inhumans as a result of the BSE epidemic in Britain and is most probablycaused by the consumption of food products derived from cattle infectedwith BSE or “mad cow disease”. An unknown number of people in the UKingested food potentially contaminated with nervous tissue fromBSE-infected cattle during the mid 1980s to early 1990s. Because theincubation period for the orally contracted disease may be more than 20years in humans, the true incidence of vCJD may not become apparent formany years. To date, over 130 people are known to have contracted thedisease, primarily in the UK; however, cases have been reported inCanada, France, Hong Kong, Ireland, Italy, and the US. The export ofcontaminated bovine feed products from the UK worldwide indicates apossible global presence of BSE and hence the probability of vCJD.Consistent with these observations is the detection of BSE in mostEuropean countries, Japan and Israel. Consequently, the ability todetect and remove infectious prion protein from a variety of materialsincluding food products is of profound importance.

[0005] Historically, the diagnosis of TSEs was based on the occurrenceof clinical signs of the disease and could be confirmed only bypost-mortem histological examination of brain tissue. A characteristicof all TSEs is the lack of a measurable host immune response to theagent. Thus, no antibodies are produced and no conventional serologictest can be used to identify infected animals. Recently, identificationof abnormal prion protein in the brain has improved the ability to makea disease diagnosis.

[0006] In addition to ingestion of infected products of bovine origin,blood transfusion and organ transplantation represent another potentialmode of transmission of vCJD among humans. The likelihood oftransmissibility of vCJD in humans by blood transfusion is currentlyunknown, but based on data from experimental animal models includingtransmission from sheep experimentally infected orally with BSE andsheep naturally infected with scrapie, appears to be a very likelypossibility. Unlike other human TSEs, PrPsc is present in thelymphoreticular system of vCJD patients, thereby increasing theprobability of the infectious agent being in blood and its transmissionthrough blood transfusion. Other factors elevating concern about therisk of transmission by transfusion include the unknown, but presumablyhigh, numbers of people exposed to BSE and lack of a preclinicaldiagnostic test for vCJD. Moreover, the virulence of vCJD appears to beenhanced following species adaptation in primates and mice, suggestingthat human to human transmission may be more efficient than cow tohuman. Thus, there is an urgent need for methods to prevent thetransmission of vCJD by blood transfusion. Such measures may includeearly identification of infected donors and their deferral, removal andinactivation of TSE agents in animal derived food and health productsintended for animal or human consumption or applications, human andbovine derived blood-derived products, and organ transplants.Unfortunately, PrPsc is remarkably resistant to chemical and physicalmethods of inactivation, and a selective method of inactivation iselusive.

[0007] Prion removal through the specific interaction with ligandsappears more promising. A number of ligands have already been identifiedthat bind to prion protein. Combinatorial peptide libraries have beenscreened for ligands that bind to the octapeptide repeat sequence(PHGGGWGQ (SEQ ID NO:220)) found in all known mammalian prion proteinsand a series of ligands were discovered, as described in PCT/US01/11150.Other materials include a variety of polymers, for example, aminopolymethacrylate from TosoBioSep, ion exchange resins generally (seeU.S. Pat. No. 5,808,011 to Gawryl et al.), ligands that interact withamyloid plaque for example, Congo Red (Ingrosso, L., et al., Congo redprolongs the incubation period in scrapie-infected hamsters. J. Virology69:506-508 (1995)), 4-iodo, 4-deoxy doxorubicin (Tagliavini, F., et al.,Effectiveness of anthracycline against experimental prion diseases inSyrian hamsters. Science 276:1119-1122 (1997)), amphotericin B,porphyrins and phthalocyanines (Priola, S. A., et al., Porphyrin andPhthalocyanine antiscrapie compounds, Science 287:1503-1506 (2000)),metals (Stockel et al., Biochemistry, 37, 7185-7193 (1998)), peptidesthat interact with PrP to form complexes (see U.S. Pat. No. 5,750,361 toPrusiner et al. and Soto, C. et al., Reversion of prion proteinconformational changes in synthetic β-sheet breaker peptides, Lancet,355:192-197 (2000)), heparin and other polysulphated polyanions(Caughey, B., et al., Binding of the Protease-sensitive form of prionprotein PrP to Sulphated Glycosaminoglycan and Congo Red, J. Virology68:2135-2141(1994)), antibodies (Kascsak, R. J., et al., Immunodiagnosisof prion disease, Immunological Invest. 26:259-268 (1997)), and otherproteins, e.g. plasminogen (Fischer, M. B. et al., Binding ofdisease-associated prion protein to plasminogen., Nature 408:479-483(2000)). Currently, no ligand has been fully characterized or found tobe able to bind to prion from a wide variety of media, although some maybe useful in specific circumstances (see U.S. Pat. No. 5,808,011 toGawryl et al.).

[0008] To date, human TSE diseases are 100% fatal. Unfortunately, eventhough a number of compounds including amphotericins, sulphatedpolyanions, Congo Red dye and anthracycline antibiotics have beenreported as prospective therapeutic agents, all have demonstrated onlymodest potential to impede prion propagation, and none have been shownto have any effect on the removal of pre-existing prions from aninfected host. Thus, there remains an urgent need for new therapeuticagents.

[0009] The assembly and disassembly of normally soluble proteins intoconformationally altered and insoluble forms are thought to be acausative process in a variety of other diseases, many of which areneurological diseases. The relationship between the onset of the diseaseand the transition from the normal to the conformationally alteredprotein is poorly understood. Examples of such insoluble proteins inaddition to prion include: β-amyloid peptide in amyloid plaques ofAlzheimer's disease and cerebral amyloid angiopathy (CAA); α-synucleindeposits in Lewy bodies of Parkinson's disease, tau in neurofibrillarytangles in frontal temporal dementia and Pick's disease; superoxidedismutase in amyotrophic lateral sclerosis; and huntingtin inHuntington's Disease.

[0010] Often these highly insoluble proteins form aggregates composed ofnon-branching fibrils with the common characteristic of a β-pleatedsheet conformation. In the central nervous system, amyloid can bepresent in cerebral and menningeal blood vessels (cerebrovasculardeposits) and in brain parenchyma (plaques). Neuropathological studiesin human and animal models indicate that cells proximal to amyloiddeposits are disturbed in their normal functions.

[0011] The precise mechanism by which neuritic plaques are formed andthe relationship of plaque formation to the disease-associatedneurodegenerative processes are largely unknown. Methodologies that canreadily separate or that can distinguish between two or more differentconformational forms of a protein, for example, PrPc and PrPsc, areneeded to understand the process of conversion and to find structuresthat will specifically interact with the disease associated form.Current methodologies for separating or distinguishing between isoformsinclude: differential mobility in polyacrylamide gels in the presence ofa chaotrope such as urea, i.e., transverse urea gradient (TUG) gels;differential sensitivity to protease treatment, for example, proteinaseK (PK) and the detection of the PK resistant digest product of PrPscreferred to a PrPres; differential temperature stability; relativesolubility in non-ionic detergents; and the ability for fibrillarstructures to bind certain chemicals, for example, Congo red andisoflavin S. However, there remains an unmet need to identify highaffinity reagents that are specific for the conformationally alteredprotein and especially forms associated with disease. Such reagentswould be useful for developing possible diagnostic kits, separation andpurification of the different forms of protein, for removal ofinfectious forms of the disease from therapeutic agents, biologicalproducts, vaccines and foodstuffs, and for therapy.

SUMMARY OF THE INVENTION

[0012] Ligands that bind to prion proteins and their applications areprovided. The ligands are peptides that bind with selectivity andspecificity to prion analytes. The ligands are capable of binding to oneor more forms of prion protein including cellular prion protein (PrPc),infectious prion protein (PrPsc), and recombinant prion protein (PrPr).Prions from various species, including humans and hamsters, are bound bythe ligands. Compositions containing the prion protein binding ligandson a support such as a resin or a membrane are also provided.

[0013] The ligands are useful for detecting or removing a prion proteinfrom a sample, such as a biological fluid or an environmental sample.The ligands are used to detect or remove all prion protein from thesample or can be selectively chosen to detect or remove a single form ofprion protein and can therefore be used to distinguish betweeninfectious and non-infectious prion protein in the sample from patientsafflicted with human TSEs and animals afflicted with scrapie, BSE andCWD.

[0014] Also provided is a method of treating or retarding thedevelopment of a prion-associated pathology in a subject. For example,the ligands of the invention may be useful in treating pathologies suchas CJD, vCJD, GSS, fatal insomnia, scrapie, BSE and CWD. Such ligandsmay act by inhibiting polymerization of PrPsc or through inhibiting theinteraction of PrPsc and PrPc thereby slowing down the development offurther PrPsc.

[0015] Another aspect of the invention provides a method for identifyingadditional ligands, particularly ligands specific for theconformationally altered forms of proteins, some of which are involvedin the development of diseases. The described methodology is alsoappropriate for the discovery, evaluation or screening of large numbersof potential drug candidates that bind directly to PrPsc.

[0016] Other features and advantages of the invention will be apparentfrom the following detailed description and preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 Chemiluminescent signals from beads from a combinatoriallibrary binding haPrPc and haPrPsc from brain homogenate. PrPc and PrPscwere detected through binding of a specific monoclonal antibody (3F4)and alkaline phosphatase conjugated secondary antibodies specific for3F4. Light produced by a chemiluminescent substrate specific foralkaline phosphatase was detected on autoradiography film. The locationsof signals generated from beads from a combinatorial library arenumbered. The ligands on the beads were subsequently sequenced. Thesebeads did not produce a signal prior to transfer and denaturation, butemitted a strong signal following transfer and denaturation of boundproteins and labeling with enzyme conjugate 3F4 antibody.

[0018]FIG. 2 Binding of huPrPc from extracts of normal human brain toaffinity resins in a column format. Brain homogenate and beads wereprepared and equilibrated in either phosphate (PBS) or citrate phosphatedextrose (CPD) buffers. The strength of the signal on the Western blotsis a function of the strength of PrPc binding to the resin. Lane 1contains molecular weight marker (MW); Lane 2, 20 μl of 0.1% normalhuman brain homogenate. Lane 3-8, PrPc eluted from beads.

[0019]FIG. 3 Binding of huPrPsc from extracts of CJD infected humanbrain to affinity resins in a batch format. The figure is a Western blotthat shows the amount of prion eluted from beads following contact witha homogenate containing huPrPsc from a patient with sporadic CJD. Thebeads were washed following contact with the homogenate that were eithertreated with PK to reveal the presence of PrPres or remained untreated.They were boiled in buffer containing SDS to release bound protein, andthe samples were resolved by SDS-PAGE followed by Western blotting. Thebinding of huPrPsc and PrPc to the resins is demonstrated by presence ofPrP specific bands following probing with a monoclonal antibody, 3F4.Peptide sequences are indicated at the top of the gel. Samples digestedwith PK are identified as (+), undigested as (−).

[0020]FIG. 4 Binding of huPrPsc from extracts of CJD infected humanbrain to affinity resins in a column format. Peptide sequences areindicated at the top of the gel. Samples previously digested with PK areidentified as + and undigested as −. Controls included 20 μl of 1% brainhomogenate. PrPc and PrPsc were specifically detected using monoclonalantibody 3F4 and visualized by detection of a chemiluminescent signal.

[0021]FIG. 5 Diagram of the “bead blot” transfer set-up. Beads arearrayed in a gel following incubation with starting materials. Boundprotein is transferred from the beads and captured on the membrane viacapillary transfer of buffer as indicated.

[0022]FIG. 6 Removal of PrPres from infected RBCCs by various affinityresins. Red Blood Cell Concentrates (RBCCs) were spiked with brainhomogenate from hamsters infected with Scrapie and passed in successionthrough columns of resins with various affinity ligands. Resin-boundproteins were analyzed by gel electrophoresis. Gel loading pattern isshown in Table 11.

DETAILED DESCRIPTION

[0023] Ligands that bind to prion proteins and their applications aredescribed herein. The ligands are proteins, peptides or polypeptidesthat bind with specificity and affinity to prion proteins. Preferably,the ligands have a molecular weight of 6 kDa or less.

[0024] The ligands are useful in methods for detecting prion protein ina sample, such as a human or animal derived biological fluid or anenvironmental sample, as well as methods for diagnosing and treatingprion disease. For example, the ligands of the invention may be usefulin treating diagnosing pathologies such as CJD, vCJD, GSS, fatalinsomnia, scrapie, BSE and CWD and other TSEs using whole blood, bloodcomponents, cells, serum, plasma, plasma derivatives, cerebrospinalfluid, urine, tears tonsils, appendix and others. The ligands may alsobe useful for the removal of prion protein from a sample, such as ablood sample, blood components, cells, serum, plasma, plasmaderivatives, cerebrospinal fluid, urine, tears tonsils, appendix andothers. The ligands are used to detect or remove all prion protein fromthe sample or can be selectively chosen to detect or remove a singleform of prion protein and can therefore be used to distinguish betweeninfectious and non-infectious prion protein in the sample.

[0025] The methods described may be used for screening polymers,synthetic compounds and libraries of synthetic compounds for additionalligands to prions.

[0026] Also provided herein is a method for identifying additionalligands, particularly ligands specific for the conformationally alteredforms of proteins, some of which are involved in the development ofdiseases.

[0027] Also provided is a methodology that is appropriate for thediscovery, evaluation or screening of large numbers of potential drugcandidates.

[0028] Definitions

[0029] The terms “a,” “an” and “the” as used herein are defined to mean“one or more” and include the plural unless the context isinappropriate.

[0030] The term “3F4” refers to the monoclonal antibody specific tonative forms of PrPc, but not native PrPsc or PrPres. The antibody hasspecificity for denatured forms of hamster and human PrPc, PrPsc andPrPres.

[0031] As used herein, the terms “blood-derived compositions” and bloodcompositions are used interchangeably and are meant to include wholeblood, red blood cell concentrate, plasma, serum, platelet rich andplatelet poor fractions, platelet concentrates, white blood cells, bloodplasma precipitates, blood plasma fractionation precipitates andsupernatants, immunoglobulin preparations including IgA, IgE, IgG andIgM, purified coagulation factor concentrates, fibrinogen concentrate,or various other compositions which are derived from humans or animals.It also includes purified blood derived proteins prepared by any ofvarious methods common in the art including ion exchange, affinity, gelpermeation, and/or hydrophobic chromatography or by differentialprecipitation.

[0032] The term “combinatorial library” refers to a collection ofchemicals that have been synthesized by solid-phase combinatorialchemistry techniques. This definition encompasses using asplit-couple-recombine method that generates millions of random peptidesof a defined length or may be designed to include defined structures.The building blocks may be natural amino acids, synthetic molecules,amino acid analogs, branched analogs, triazine dyes, and the like.

[0033] The term “conservative variations” or “conservative modifiedvariations” of a particular sequence refers to amino acids or otherclosely related structures that have substantial chemical similarity.Furthermore, individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids in an encoded sequence are conservatively modified variationswhere the alterations result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.The following six groups each contain natural amino acids that areconservative substitutions for one another:

[0034] 1) Serine (S), Threonine (T);

[0035] 2) Aspartic acid (D), Glutamic acid (E);

[0036] 3) Asparagine (N), Glutamine (Q);

[0037] 4) Arginine (R), Lysine (K);

[0038] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V),Alanine (A)

[0039] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0040] Numerous unnatural amino acids are also considered conservativesubstitutions of naturally occurring amino acids. Two polypeptides aresaid to be “identical” if the sequence of amino acid residues in the twosequences is the same when aligned for maximum correspondence. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482(1981), by the homology alignment algorithm of Needleman and Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection.

[0041] The term “ligand” refers to a molecule to which a protein,peptide or polypeptide binds. The ligands of the present invention canbe antibody preparations, proteins, peptides, polypeptides, amino acids,nucleic acids, carbohydrates, sugars, lipids, organic molecules,polymers, and/or putative therapeutic agents, and the like.

[0042] The terms “protein” “peptide,” “polypeptide” and “oligopeptide”are used interchangeably and are defined herein as a chain of aminoacids in which carbons are linked through peptide bonds formed by acondensation reaction between the carboxyl group of one amino acid andthe amino group of another amino acid. The terminal amino acid at oneend of the chain (i.e., the amino terminal) has a free amino group,while the terminal amino acid at the other end of the chain (i.e., thecarboxy terminal) has a free carboxyl group. As such, the term “aminoterminus” (abbreviated N-terminus) refers to the free amino group on theamino acid at the amino terminal of the peptide, or to the amino group(imino group when participating in a peptide bond) of an amino acid atany other location within the peptide. Similarly, the term “carboxyterminus” (abbreviated C-terminus) refers to the free carboxyl group onthe amino acid at the carboxy terminus of a peptide, or to the carboxylgroup of an amino acid at any other location within the peptide. Whensynthesized on resin by Merrifield synthesis the C-terminal carboxylgroup is coupled to the resin usually through a peptide bond to animmobilized amino group.

[0043] Typically, the amino acids making up a peptide are numbered inorder, starting at the amino terminal and increasing in the direction ofthe carboxy terminal of the peptide. Thus, when one amino acid is saidto “follow” another, that amino acid is positioned closer to the carboxyterminal of the peptide than the “preceding” amino acid.

[0044] The term “PrPc” refers to the native prion protein molecule whichis naturally and widely expressed within the body of the Mammalia. Itsstructure is highly conserved and is not associated with a diseasestate.

[0045] The term “PrPsc” refers to the conformationally altered form ofthe PrPc molecule that is that is thought to be infectious and isassociated with TSE/prion diseases, including vCJD, CJD, kuru, fatalinsomnia, GSS, scrapie, BSE, CWD, and other rare TSEs of captive andexperimental animals. It has the same amino acid sequence as normal,cellular PrPc, but has converted some of the α-helix to β-sheet and isassociated with a disease state.

[0046] The term “PrPres” refers to the proteinase resistant derivativesof the PrPsc protein of 27-30 kDa that remain following partialdigestion of PrPsc with PK.

[0047] The term “PrPr” refers to the prion protein expressed byrecombinant technology.

[0048] The term “PrP” refers to prion protein in general.

[0049] The term “residue” is used herein to refer to an amino acid (D orL) or an amino acid mimetic that is incorporated into an oligopeptide byan amide bond or an amide bond mimetic. As such, the amino acid may be anaturally occurring amino acid or, unless otherwise limited, mayencompass known analogs of natural amino acids that function in a mannersimilar to the naturally occurring amino acids (i.e., amino acidmimetics). Moreover, an amide bond mimetic includes peptide backbonemodifications well known to those skilled in the art.

[0050] The term “substantial identity” means that a polypeptidecomprises a sequence that has at least 66% or more amino acids incommon. Another indication that polypeptide sequences are substantiallyidentical is if one peptide is immunologically reactive with antibodiesraised against the disclosed peptide. Thus, the peptides of theinvention include peptides and other chemicals immunologically reactivewith antibodies raised against the disclosed immunogenic peptides.

[0051] The term “capable of binding” as used herein refers to binding oftwo or molecules to form a complex with each other, for example, bindingof a ligand to a protein or a peptide, under conditions, wherein the twoor more molecules are capable of forming a complex, such as aprotein-ligand complex.

[0052] Ligands that Bind to a Particular Amino Acid Sequence of PrP

[0053] The prion-binding ligands described herein are all smallmolecules, preferably peptides. The ligands bind to peptides,polypeptides derived from the prion protein, or the entire prionmolecule. As used herein, no particular length is implied by the term“peptide.” Preferably, the ligands described herein bind to a prionprotein having one or more of the following amino acid sequences:

[0054] RYPxQ (SEQ ID NO:221), wherein x is G, P or N

[0055] XxYYux (SEQ ID NO:222), wherein x is any amino acid, and u is Ror Q

[0056] More preferably the ligands bind to a prion protein having one ormore of the following amino acid sequences: RYPGQ (SEQ ID NO:1) DRYYRD(SEQ ID NO:2) QAYYQR (SEQ ID NO:3) QVYYRP (SEQ ID NO:4)

[0057] Labelled peptides having one or more of the amino acid sequencesprovided above are useful when used to probe combinatorial libraries forligands that bind to prions. Preferably, the peptides are radiolabelledand acetylated at the amino terminus and amidated at the carboxyterminus when used to screen libraries for prion ligands.

[0058] The amino acid sequence of the ligands described herein lack theamino acid sequences disclosed in WO 01/77687, which binds to theoctapeptide repeat sequence of the prion protein.

[0059] In a first preferred embodiment, the ligand is a protein orpeptide having an amino acid sequence that binds to SEQ ID NO:1. Theamino acid sequences set forth in Table 1 below (SEQ ID NOS. 5-13) areexamples of amino acid sequences that bind to SEQ ID NO:1. Therefore,ligands having one or more of the sequences set forth in Table 1 areincluded in the ligands of the first preferred embodiment. The aminoacid sequences set forth in Table 1 were identified in a 6-mer libraryscreened for 6-mers that bind to SEQ ID NO:1. The library wasconstructed with an alanine (A) as a spacer between the resin and thecombinatorial peptides of the library and is represented as the final Ain the sequences which is included in Table 1. It will be understood bythose skilled in the art that the ligands provided herein are notlimited to those having the exemplary sequences set forth in Table 1.TABLE 1 Six-amino acid sequences binding to SEQ ID NO: 1 SEQ ID NOSEQUENCE 5 KIHKFLA 6 GTHDFQA 7 KFGSTHA 8 FVNEIEA 9 GLHFKSA 10 GRVLHHA 11QKNSEWA 12 HAYFTHA 13 WPKGAVA

[0060] In a second preferred embodiment, the ligand is a protein orpeptide having an amino acid sequence that binds to SEQ ID NO:2. Theamino acid sequences set forth in Table 2 below (SEQ ID NOS:14-22) areexamples of amino acid sequences that bind to SEQ ID NO: 2. Therefore,ligands having one or more of the sequences set forth in Table 2 areincluded in the ligands of the second preferred embodiment. The aminoacid sequences set forth in Table 2 were identified in a 6-mer libraryscreened for 6-mers that bind to SEQ ID NO:2. The library wasconstructed with an alanine (A) as a spacer between the resin and thecombinatorial peptides of the library and is represented as the final Ain the sequences. The amino acid lysine (K) is present eleven times, andthe amino acid histidine (H) is present seven times, both which areabove an average distribution of three. Therefore, six-mers containingthe amino acid lysine (K) or histidine (H) are preferred. It will beunderstood by those skilled in the art that the ligands provided hereinare not limited to those having the exemplary sequences set forth inTable 2. TABLE 2 Six-amino acid sequences binding to SEQ ID NO: 2 SEQ IDNO SEQUENCE 14 RPWKKAA 15 PKHIWPA 16 HKLWGVA 17 GGYKPYA 18 ENVSQNA 19HTYYNGA 20 KKKSDHA 21 HHLKGTA 22 KKHGVWA

[0061] In a third preferred embodiment, the ligand is a protein orpeptide having an amino acid sequence that binds to SEQ ID NO:3. Theamino acid sequences set forth in Table 3 below (SEQ ID NOS:23-31) areexamples of amino acid sequences that bind to SEQ ID NO: 3. Therefore,ligands having one or more of the sequences set forth in Table 3 areincluded in the ligands of the third preferred embodiment. The aminoacid sequences set forth in Table 3 were identified in a 6-mer libraryscreened for 6-mers that bind to SEQ ID NO: 3. The library wasconstructed with an alanine (A) as a spacer between the resin and thecombinatorial peptides of the library and is represented as the final Ain the sequences. In cases of sequence ambiguity in identification, oneor more amino acids are given in a single position in the Table, forexample, (A/G) as shown in SEQ ID NO: 29. The amino acid histidine (H)appears 10 times in these sequences, is found in six of the eightpeptides, and is well above an average distribution of three. Allpeptides except SEQ ID NO: 23 have a net positive charge at pH 7.Therefore, six-mers containing the amino acid histidine (H) and peptideshaving a net positive charge at pH 7 are preferred. It will beunderstood by those skilled in the art that the ligands provided hereinare not limited to those having the exemplary sequences set forth inTable 3. TABLE 3 Six-amino acid sequences binding to SEQ ID NO: 3 SEQ IDNO SEQUENCE 23 DGTQAHA 24 APHRNNA 25 HHGHNIA 26 HTWHGQA 27 HVFVTWA 28THHFYIA 29 KLGWG(A/G)A 30 GSKKKEA

[0062] In a fourth preferred embodiment, the ligand is a protein orpeptide having an amino acid sequence that binds to SEQ ID NO:4. Theamino acid sequences set forth in Table 4 below (SEQ ID NOS:31-47) areexamples of amino acid sequences that bind to SEQ ID NO:4. Therefore,ligands having one or more of the sequences set forth in Table 4 areincluded in the ligands of the fourth preferred embodiment. The aminoacid sequences set forth in Table 4 were identified in a 6-mer libraryscreened for 6-mers that bind to SEQ ID NO:4. The library wasconstructed with an alanine (A) as a spacer between the resin and thecombinatorial peptides of the library and is represented as the final Ain the sequences. It will be understood by those skilled in the art thatthe ligands provided herein are not limited to those having theexemplary sequences set forth in this Table 4. In cases of sequenceambiguity in identification, one or more amino acids are given in asingle position, for example, (W/G) as shown in SEQ ID NO:33. The aminoacid in the second position of SEQ ID NO:37 could not be positivelyidentified. The sequence “LL” (two leucines) appears in SEQ ID NOS:31,32, 41, 43 and 45 and its close analogs LI, VL, II (isoleucine orvaline) appear in SEQ ID NOS:33, 36, 38, 40 and 44. LL does not appearin any other screens for prion-derived peptides or proteins. Inaddition, 15 of 17 peptides contain an aromatic amino acid, such asphenylalanine, tryptophan or tyrosine (F, W or Y). Seven peptidesequences are neutrally charged, but have a positive terminal aminogroup. Therefore, six-mers containing one or more leucine (L) or leucineanalogs, such as isoleucine or valine (I or V) in sequence, preferablyLL, LI, VL or II; six-mers containing an aromatic amino acid, such asphenylalanine, tryptophan or tyrosine (F, W or Y); and six-mers that areneutrally charged, but having a positive terminal amino group arepreferred. TABLE 4 Six-amino acid sequences binding to SEQ ID NO: 4 SEQID NO SEQUENCE 31 PLLVVWA 32 WLLVGGA 33 (W/G)QVLVYA 34 RRHQRQA 35LPWTFGA 36 IFIIITA 37 P(X)IEPHA 38 EWGIIWA 39 GWYIYFA 40 TLILFHA 41FLLSNHA 42 WQIRFFA 43 VLLVFEA 44 GWVLEIA 45 FLLIDTA 46 GFLFKFA 47PWTIYIA

[0063] Ligands that Bind to Hamster PrPc

[0064] In another embodiment, the ligand is a peptide that binds withspecificity and selectivity to one or more forms of prion protein foundin a particular species, such as human or another mammal, such as ahamster. Exemplary ligands that bind to prion protein (PrPc) havingdifferent amino acid sequence lengths, two-mer, three-mers, four-mers,five-mers and six-mers, preferably having a molecular weight of 6 kDa orless, are provided below.

[0065] Exemplary two-mer ligands that bind to native prion in hamsters(haPrPc) are set forth in SEQ ID NOS:48-50, which are listed in Table5A. The ligand preferably contains the amino acid tryptophan (W). Thepreferred ligand is neutrally charged, but has a positive chargedterminal amino acid group at pH 7. The preferred ligand is a two-mercontaining the tryptophan (W). Substitution of naphthyl-alanine (na) fortryptophan also resulted in binding of PrP in these sequences. Thelibrary was synthesized directly onto the resin (a Toyopearl amino resinTosoh Bioscience LLC, Monctgomerville, Pa.) without a spacer. SEQ IDNO:50 was found twice (2×) in the screens. TABLE 5A Two-amino acidsequences binding to haPrPc SEQ ID NO SEQUENCE 48 WH 49 WW 50 LW (2×)

[0066] Exemplary three-mer ligands that bind to prion in hamsters(haPrPc) are set forth in SEQ ID NOS:51-61, which are listed in Table5B. An aromatic amino acid, F, W or Y appears in all peptides selectedexcept SEQ ID NO:60. The amino acid A appears three times at theposition closest to the resin and was used as a spacer between resin andpeptide library in the some libraries. Neither R nor K is present, but Eappears three times providing a negative charge to three of eightsequences. TABLE 5B Three-amino acid sequences binding to haPrPc SEQ IDNO SEQUENCE 51 WNA 52 EFW 53 LPW 54 YEY 55 WPA 56 FNQ 57 YHE 58 LFA 59NHY 60 TLG 61 WVD

[0067] Exemplary four-mer ligands that bind to prion in hamsters(haPrPc) are set forth in SEQ ID NOS:62-64, which are listed in Table5C. The library was constructed with an alanine spacer between the resinand the combinatorial peptide and is present in the sequences below atthe last position. An aromatic amino acid appears in the first positionof all peptides selected. In addition, all peptides selected contain anacidic amino acid (D or E) at the third or fourth position. WXD appearsonce, where X is any amino acid. TABLE 5C Four-amino acid sequencesbinding to haPrPc SEQ ID NO SEQUENCE 62 YWDQA 63 YVHEA 64 WFDEA

[0068] Exemplary five-mer ligands that bind to prion in hamsters(haPrPc) are set forth in SEQ ID NOS: 65-68, which are listed in Table5D. An aromatic amino acid and an acidic amino acid appear in allpeptides selected. D or E are present in position 4 or 5 of all ligands.The sequence WXD appears in SEQ ID NO:65, 67 and 68. TABLE 5D Five-aminoacid sequences binding to haPrPc SEQ ID NO SEQUENCE 65 LQWYDA 66 YTHSEA67 WIDYEA 68 VWIDAA

[0069] Exemplary six-mer ligands that bind to prion in hamsters (haPrPc)are set forth in SEQ ID NOS:69-100, which are listed in Table 5E. Thelibrary was constructed with an alanine spacer between the resin and thecombinatorial peptide and is present in the sequences below at the lastposition. An aromatic amino acid, F, W or Y appears in most (29 of 32)peptides selected as do D or E (29 of 32). In addition, 20 peptides havetwo aromatic amino acids in their sequence. The consensus sequence “WXD”appears in SEQ ID NOS:75, 79, 83, 86 and 89. A sequence containing an(F/W/Y)X(D/E)(F/W/Y) SEQ ID NO:)) appears in SEQ ID NOS:71, 73, 77, 78,91 and 95 and (F/W/Y)(D/E)X(F/W/Y) SEQ ID NO:)) appears in SEQ IDNOS:70, 72, 82, 91 and 95. Twenty four of 32 peptides have an aromaticamino acid plus an acid group in positions 1-3; 23 have a net negativecharge in positions 4-6. Twenty peptides have both an aromatic aminoacid plus an acid amino acid in positions 1-3 and are also net negativein positions 4-6. TABLE 5E Six-amino acid sequences binding to haPrPcSEQ ID NO SEQUENCE 69 WDEAEEA 70 YDSYDDA 71 NDFIDFA 72 YEPWGSA 73EYGDWWA 74 WDYDQEA 75 DWGDPFA 76 DWPEVWA 77 FHDFSEA 78 DTFWDYA 79WNDLDNA 80 ASALVYA 81 LINAGGA 82 WESYVTA 83 WSDEGYA 84 YRWTGPA 85YEDQWQA 86 EWADDNA 87 YEIDYGA 88 EFGYFDA 89 WGDEQDA 90 HEEDWAA 91FEDFELA 92 TWGIDEA 93 WDPTDYA 94 NDKIHTA 95 FEDFFSA 96 YEWAEQA 97THVYFLA 98 (S/T/W)XDFSDA 99 YRTPNEA 100 (G/L)RSETA

[0070] Ligands that Bind to Hamster PrPc and Hamster PrPsc

[0071] In another embodiment, the ligand is a peptide that binds withspecificity and selectively to two or more forms of prion. Ligands thatbind to both (PrPc) and/or conformationally changed (PrPsc) prionprotein are provided below. Exemplary three-mer ligands that bind toprion in hamsters (haPrPc) are set forth in SEQ ID NOS:101-115, whichare listed in Table 6. An aromatic amino acid appears in most (15 of 18)peptides selected as do D or E (15 of 18). In addition, seven peptideshave two aromatic structures and an acidic amino acid. The sequence WXDappears in SEQ ID NOS:105 and 115. Structures selected to bindpreferentially PrPsc over PrPc are SEQ ID NO:111 and SEQ ID NO:114, bothhaving R at position 3. SEQ ID NO:101-115 were identified in a 3-merlibrary to bind haPrPc and/or PrPsc from homogenates of scrapie-infectedbrain either alone (*), or mixed with normal hamster brain. TABLE 6Three-amino acid sequences binding to haPrPc and haPrPsc Light signalafter Bead color denaturation (strong (red shows strong PrPc showsstrong PrPc SEQ ID NO Sequence binding) and/or PrPsc binding)  52 EFW*Bright pink Strong  54 YEY Pink 101 IHN Light pink 102 WEY Bright pink103 DYW Pink 104 WDW Pink 105 WQD Pink 106 YFE Pink 106 YFE* Red Strong107 NYE Pink 108 SYA Light pink None 109 WDL Bright pink Strong 110 WLEBright pink Weak 111 VQR Bright pink Very strong 112 YID* Bright pinkStrong 113 RWD* Bright pink Strong 114 DVR* White Strong 115 WSD* RedStrong

[0072] Ligands that Bind to Human PrPc

[0073] Ligands that bind to (huPrPc) prion protein are provided below.Exemplary three-mer ligands that bind to prion in humans (haPrPc) areset forth in SEQ ID NOS:116-139, which are listed in Tables 7A and B. Ofthe trimer sequences (Table 7A) W/YXD appears in four of the six trimersequences and five of the six have a hydrophobic and an acidic aminoacid residue. TABLE 7A Three-amino acid sequences binding to huPrPc SEQID NO SEQUENCE 116 HWD 117 WQD 118 WDD 119 WED 120 ITN 121 YED

[0074] The six-mer library was constructed with an alanine spacerbetween the resin and the combinatorial peptide and is present in thesequences below at the last position (Table 7B). Amino acids F, W or Yappear in 13 of 18 6-mer peptides, and D or E in 17 of 18 peptides. Sixpeptides have an aromatic and an acid amino acid in positions 1-3 andare also net negative in positions 4-6. In addition, five peptides havetwo aromatic structures and an acid amino acid. WxD is present in SEQ IDNO:124 and (F/W/Y)x(D/E)(F/W/Y) (SEQ ID NO:223) is present in SEQ IDNOS:124 and 133. Excluding the N-terminal amino charge the majority ofsequences are net negative and only SEQ ID NO:139 carries a partial netpositive at neutral pH. SEQ ID. NOS: 116-121 were identified in a 3-merlibrary to bind huPrPc from normal human brain homogenates. SEQ IDNOS:122-139 were identified in a 6-mer library to bind either humanplatelet poor plasma or platelet rich plasma (*) derived huPrPc. TABLE7B Six-amino acid sequences binding to huPrPc SEQ ID NO SEQUENCE 122RVADEEA 123 EYYVDAA 124 WQDFNLA 125 YDNPIDA 126 YFNEHEA 127 EWGADGA 128DVIYSHA 129 WHILEEA* 130 NPHENFA* 131 HEDNGGA 132 SDSEGPA 133 EFQEFTA134 QEGDEIA 135 DIYAETA 136 DRVRETA 137 FEEPQWA* 138 FEGEEFA* 139(T/L)FNIHA*

[0075] Ligands the Bind to Human Recombinant PrP

[0076] Ligands that bind to recombinant (PrPr) prion protein areprovided below. Exemplary three-mer ligands that bind to recombinantprion in humans (huPrPr) are set forth in SEQ ID NOS:54, 105, 140-153,which are listed in Table 8. Amino Acids W, F or Y appear in all 16peptides selected from D or E in 13 of 16 peptides. The consensussequence WXD appears in SEQ ID NOS:105, 143 and 145. Some peptides havebeen previously identified to bind PrPc and SEQ ID NOS:149 and 153 wereidentified twice in this screen. SEQ ID NOS:54 105, 140-153, wereidentified in a 3-mer library to bind huPrPr (Prionics AG, Switzerland,Cat.) #03-0404) diluted into (*) 0.5% sarcosyl or (**) PBS. In Table 8,2.5 mg of dry weight of resin from a combinatorial library per columnwas exposed to 0.5 μg/ml of PrPr diluted into 0.5% sarcosyl (*) or intophosphate-buffered saline (**) containing 1% BSA. Sequences found twicein the screen are denoted 2×. TABLE 8 Three-amino acid sequences bindingto huPrPr SEQ ID NO SEQUENCE  54 YEY** 105 WQD* and **(2×) 140 YDW* 141NYT* 142 SYT* 143 WAD* 144 QWG* 145 WGD* 146 EYF* 147 WEH* 148 LYD* 149DYY* *(2×) 150 FYE** 151 EYY** 152 YDY** 153 WDH** (2×)

[0077] Six-Mer Ligands that Bind to Human PrPc, Human PrPsc or Both

[0078] Six-mer ligands that bind to (PrPc) prion protein,conformationally changed (PrPsc) prion protein, or both are provided inTABLE 9A. The six-mer library was constructed with an alanine spacerbetween the resin and the combinatorial peptide and is present in thesequences below at the last position The ligands may preferentially tohuPrPsc. Exemplary ligands are set forth in SEQ ID NOS:154-173, whichare listed in Table 9A. All ligands except SEQ ID NO:156 contained anaromatic amino acid and 15 of 20 contained an acidic amino acid. Thosewith greater specificity for huPrPsc over PrPc are SEQ ID NO:154, 155and 156. Detection of ligands with increased specificity for PrPsc in abrain homogenate derived from a sporadic CJD patient was obtainedthrough selective proteolysis of PrPc prior to transfer of protein frombeads to membrane. This library included the unnatural aromatic aminoacid 2-naphthyl-alanine (na). TABLE 9A Six-amino acid sequences thatbind to huPrPc, huPrPsc or both SEQ IN NO Sequence 154* RES(na)NVA 155*ES(na)PRQA 156* VARENIA 157* RWEREDA 158** EWWETV 159** SVYQLDA 160**(na)HEFYGA 161** HE(na)(na)LVA 162** A(na)VPV(na)A 163** YFDYWLA 164**FE(na)HRQA 165** WRHEPAA 166*** SS(na)KKDA 167*** R(na)DKEAA 168****(na)HEIFPA 169**** KWYHHRA 170**** HWWPHNA 171**** HWQVFYA 172****FHE(na)EIA 173**** HADF(na)QA

[0079] Ligands that Bind to Human PrPsc

[0080] Ligands that bind to conformationally changed prion protein(PrPsc) are provided below. The six-mer libraries were constructed withan alanine spacer between the resin and the combinatorial peptide andare included in the sequences below at the last position. Exemplaryligands are set forth in SEQ ID NOS:174-194, which are listed in Table9B. SEQ ID NOS:188, 189, 190, and 191 all showed highest differentiationof signal (white color and strong light signal). SEQ ID NOS:174-194 wereidentified in a 6-mer-library to bind huPrPsc from sporadic CJD brainhomogenate spiked into human plasma. Beads with ligands with highestspecificity for PrPsc were white on staining for PrPc, but produced astrong chemiluminescent signal following denaturation. TABLE 9BSix-amino acid sequences that bind to huPrPsc SEQ ID NO Sequence 174*ALHFETA 175* DDPTGFA 176* VAPGLGA 177* IFRLIEA 178* GLERPEA 179* IVVRLWA180* WHNPHYA 181* LIYKSDA 182** EKPIFNA 183** HWSEPAA 184** GHNWKEA185** YWHHDDA 186** GYPKENA 187** PVYWLYA 188*** FGEHTPA 189*** FQGTREA190*** TGTNRYA 191*** KWATRYA 192*** NSTKFDA 193*** LIYKEEA 194***EHATYRA

[0081] Three-Mer Ligands that Bind to Human PrPc, Human PrPsc or Both

[0082] Three-mer ligands that bind to (huPrPc) prion protein,conformationally changed prion protein (PrPsc), or both, are providedbelow. The ligands may bind preferentially to huPrPsc. Exemplary ligandsare set forth in SEQ ID NOS:195-212, which are listed in Table 9C. Inthis screen, the sporadic CJD brain homogenate was diluted in CPD andwas used as the source of huPrPsc. HYD was discovered 3 times in thisscreen. Red beads signified the binding of PrPc; 8 of 13 sequencescontained H. Amino Acids F, W or Y were found in all 13, and R or Kappeared only once. Three of five beads that preferentially bound PrPsc(strong signal) relative to PrPc (white bead) contained K or R. WXDappeared in SEQ ID NOS:200 and 208. SEQ ID NOS:195-212 were identifiedin a 3-mer library to bind huPrPc and/or huPrPsc treated with PK. TABLE9C Three-amino acid sequences that bind to huPrPc, huPrPsc, or bothPK-resistant SEQ ID NO Sequence 195* HND 196* HER 197* HGD 198* HSD 199*HFD 200**** WND 201**** YEH 202**** HWD 203**** YHD 204**** YDW 205****WDY 206** HYD (3×) 207** HWD 208** WTD 209*** FPK 210*** HWK 211*** WEE212*** LLR

[0083] Three-Mer Ligands that Bind to Human PrPc, Human PrPsc or Both

[0084] Three-mer ligands that bind to (PrPc) prion protein,conformationally changed (PrPsc) prion protein, or both, are providedbelow. The ligands may preferentially bind to huPrPsc. Exemplary ligandsare set forth in SEQ ID NOS:147, 152, 206, 213, and 214, which arelisted in Table 9D. These sequences were identified in a 3-mer libraryto bind huPrPsc and/or huPrPc from sporadic CJD brain homogenate dilutedinto (*) CPD buffer or (**) PBS. TABLE 9D Three-amino acid sequencesthat bind to huPrPc, huPrPsc, or both SEQ ID NO Sequence 147* WEH 152*YDY 206* HYD 213** SYF 214** EYY

[0085] Synthesis of Ligands

[0086] The ligands described herein may be produced by chemicalsynthesis. A variety of protein synthesis methods are common in the art,including synthesis using a peptide synthesizer. See, for example,Peptide Chemistry, A Practical Textbook, Bodasnsky, Ed. Springer-Verlag,1988; Merrifield, Science 232: 241-247 (1986). Preferably, the peptidesare synthesized, purified and then coupled to a resin or a membrane usedfor screening. Alternatively, the peptides are synthesized directly on aresin, and the resin-bound peptides are then purified.

[0087] The peptides are purified so that they are substantially free ofchemical precursors or other chemicals used in standard peptidepurification techniques. The language “substantially free of chemicalprecursors or other chemicals” includes preparations of a peptide inwhich the peptide is separated from chemical precursors or otherchemicals that are involved in the synthesis of the peptide.

[0088] Chemical synthesis of peptides facilitates the incorporation ofmodified or unnatural amino acids, including D-amino acids and othersmall organic molecules. Replacement of one or more L-amino acids in apeptide with the corresponding D-amino acid isoform can be used toincrease the resistance of the peptides to enzymatic hydrolysis, and toenhance one or more properties of the active peptides, such as prion orligand binding. The prion peptide and the peptide ligands describedherein can be polymers of L- or D-amino acids, or a combination of both.Also included are ligands in which analogs of the peptide ligandsdescribed herein are present in non-peptidyl linkages.

[0089] For example, in various embodiments, the peptide ligands areD-retro-inverso isomer peptides. The term “retro-inverso isomer” refersto an isomer of a linear peptide in which the direction of the sequenceis reversed and the chiralty of each amino acid residue is inverted.See, for example, Jameson et al., Nature, 368: 744-746 (1994). The netresult of combining D-enantiomers and reverse synthesis is that thepositions of carbonyl and amino groups in each amide bond are exchanged,while the position of the side-chain groups at each α-carbon ispreserved. Unless stated otherwise, it is presumed that any givenL-amino acid sequence of the invention may be made into aD-retro-inverso isomer peptide.

[0090] Additional covalent cross-links can be introduced into thepeptide sequence to constrain the structure of the peptide backbone.This strategy can be used to develop peptide analogs with increasedpotency, selectivity and stability. Macrocyclization is oftenaccomplished by forming an amide bond between peptide N- and C-termini,between a side chain and the N- or C-terminus, for example, withK3Fe(CN)6 at pH 8.5 (Samson et al., Endocrinology, 137: 5182-5185(1996)) or between two amino acid side chains. See, for example,DeGrado, Adv. Protein Chem., 39:51-124 (1988).

[0091] A number of other methods can also introduce conformationalconstraints into peptide sequences in order to improve their potency,stability and selectivity. These include the use of C α-methylaminoacids(see, for example, Rose et al., Adv. Protein Chem., 37:1-109 (1985)) orN α-methylamino acids (see, for example, Aubry et al., Int. J. Pept.Protein Res., 18:195-202 (1981)).

[0092] If desired, two or more peptide ligands can be present inmultiple copies. Identical copies of one or more peptides can be present(for example, homodimers, homotrimers, etc), or multiple copies ofpeptides varying in sequence can be present (for example, heterodimers,heterotrimers, etc.).

[0093] In an alternative, the ligands are synthesized using recombinantnucleic acid methodology. Generally, this involves creating a nucleicacid sequence that encodes the ligands, placing the nucleic acid in anexpression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if required, renaturing the protein. Techniques sufficient to guide oneof skill through such procedures are known to those skilled in the art.

[0094] Once expressed, recombinant ligands can be purified according tostandard procedures, including ammonium sulfate precipitation, affinitycolumns, column chromatography, gel electrophoresis and the like.Substantially pure compositions of about 50 to 95% homogeneity arepreferred, and approximately 80 to 95% or greater homogeneity is mostpreferred for use as therapeutic agents.

[0095] Optionally, the ligands are combined into mosaic proteins.Typically, 2 to 20 of the ligands are fused into a single polypeptide byrecombinant or synthetic techniques.

[0096] In recombinant procedures, mosaic proteins are made by ligatingsynthetic or recombinant nucleic acids which encode immunogenicpeptides. These nucleic acids can be ligated enzymatically (for example,using a DNA Ligase enzyme) or synthetically. Alternatively, a singlenucleic acid can be synthesized which encodes multiple ligand peptides.In either case, the resulting nucleic acid encodes multiple ligands, allin the same reading frame. Thus, the translated polypeptide comprisesprion-binding ligands.

[0097] Where the ligands are made by automated chemical syntheticprocedures, concatamers of peptides can be coupled directly. This isperformed chemically by joining peptides using standard chemicalmethods. Alternatively, a polypeptide can be synthetically producedwhich encodes multiple ligand peptides.

[0098] Ligand Identification

[0099] In addition to the ligands set forth above in the tables,additional ligands can be identified as follows. Peptide libraries aresynthesized and screened for the ability to bind to prion analytes. Theligands can be of any length. However, lengths from two to six aminoacids are preferred. The synthetic peptides are immobilized on beads,and the beads packed into a chromatography column. Prion analyte is thenpassed through the column and bound analyte is detected usingconventional methods such as by a labelled antibody specific for prionprotein. Beads to which the analyte has bound are identified as beingsuitable ligands.

[0100] Use of Ligands to Remove Prions

[0101] Ligands that bind prions or fragments of prions are useful for avariety of analytical, preparative, and diagnostic applications.Prion-binding ligands may be immobilized on a support such as a bead ormembrane, and used to bind and remove prion from a sample. The solidphase to which the ligands are bound is allowed to contact the sample,such as a biological fluid, under conditions sufficient to causeformation of a prion-ligand composite, and prion protein in the samplebinds to the ligand. The solid phase is then separated from the sample,thereby removing the prion protein bound to the ligand, which isattached to the solid phase, from the sample. For example, resins andmembranes for removal of contaminants are well known in the art such asthose described in U.S. Pat. No. 5,834,318 to Baumbach et al. andPCT/US01/11150.

[0102] Examples of biological samples include, but are not limited to,blood, blood-derived compositions or serum. Additional biologicalsamples include cerebrospinal fluid, urine, saliva, milk, ductal fluid,tears or semen. Other samples may contain collagen, brain and glandextracts.

[0103] Many methods for immobilizing molecules to a variety of solidsurfaces are known in the art. For instance, the solid surface may be amembrane (for example, nitrocellulose), a microtiter dish (for example,PVC, polypropylene, or polystyrene), a test tube (glass or plastic), adipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, and thelike), a microcentrifuge tube, or a glass, silica, plastic, metallic orpolymer bead. The desired component may be covalently bound, ornoncovalently attached through nonspecific bonding.

[0104] A wide variety of organic and inorganic polymers, both naturaland synthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, polyacrylate,poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate),polyvinylidene difluoride (PVDF), silicones, polyformaldehyde,cellulose, cellulose acetate, nitrocellulose, and the like. Othermaterials which may be employed, include paper, glasses, ceramics,metals, metalloids, semiconductive materials, cements or the like. Inaddition, substances that form gels, such as proteins (for example,gelatins), lipopolysaccharides, silicates, agarose and polyacrylamidescan be used. Polymers which form several aqueous phases, such asdextrans, polyalkylene glycols or surfactants, such as phospholipids,long chain (12-24 carbon atoms) alkyl ammonium salts and the like arealso suitable. Where the solid surface is porous, various pore sizes maybe employed depending upon the nature of the system. In addition, thepeptide may be incorporated during polymerization of the solid surface.

[0105] In preparing the surface, a plurality of different materials maybe employed, for example, laminates, to obtain various properties. Forexample, protein coatings, such as gelatin can be used to avoid nonspecific binding, simplify covalent conjugation, and enhance signaldetection or the like.

[0106] If covalent bonding between a compound and the surface isdesired, the surface will usually be polyfunctional or be capable ofbeing polyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature.

[0107] Prion proteins may also be separated from other proteins in asample by using affinity chromatography. Ligands according to theinvention can be attached to a solid support, such as a resin or amembrane, and used to bind and remove the prion from solution. In thisinstance, the ligand may be coupled to a solid support, for example, aninert support such as a membrane or a resin, and the prion protein bindsto the immobilized agent. The immobilized agent/prion may be detected bymeans of antibodies. If desired, one or more of the sequences obtainedfrom the initial screening may be immobilized on a resin, such apolymethacrylate or agarose. Other types of resin that may be usedinclude, for example, sepharose, cross-linked agarose, compositecross-linked polysaccharides, celite, PVDF, acrylate, polystyrene andcellulose. Membranes, such as, nylon and cellulose, may also be used.The resin may be a polymethacrylate resin.

[0108] Use of Ligands to Detect Prions

[0109] The ligands described herein are also useful in a method ofdetecting the presence of or quantifying a prion protein in a biologicalsample. A biological sample such as, but not limited to, those listedabove, is contacted with a ligand under conditions sufficient to causeformation of a complex between the prion protein and the ligand. Thecomplex is then detected by conventional methods, thereby detecting thepresence of the prion in the biological sample.

[0110] The complex is detected by labelling the ligand, combining thelabelled ligand with the sample, and detecting labelled ligand-prioncomplex. The ligand is labelled during ligand production, such as duringpeptide synthesis, or a label is conjugated to the ligand by joining itto the ligand, either covalently or non-covalently. Alternatively, abinding molecule specific for the ligand, such as an antibody, islabelled and the complex is detected indirectly. A wide variety oflabels and conjugation techniques are known and are reported extensivelyin both the scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

[0111] Detection may proceed by any known method, such asimmunoblotting, Western analysis, gel-mobility shift assays, fluorescentin situ hybridization analysis (FISH), tracking of radioactive orbioluminescent markers, nuclear magnetic resonance, electronparamagnetic resonance, stopped-flow spectroscopy, columnchromatography, capillary electrophoresis, or other methods which tracka molecule based upon an alteration in size or charge or both. Theparticular label or detectable group used in the assay is not a criticalaspect of the invention. The detectable group can be any material havinga detectable physical or chemical property. Such detectable labels havebeen well-developed and, in general, any label useful in such methodscan be applied to the present method. Thus, a label is any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include fluorescent dyes (for example, fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (forexample, ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (for example, LacZ, CAT,horse radish peroxidase, alkaline phosphatase and others, commonly usedas detectable enzymes, either in an EIA or in an ELISA), andcolorimetric labels such as colloidal gold or colored glass or plastic(for example, polystyrene, polypropylene, latex, etc.) beads. The labelmay be coupled directly or indirectly to the desired component of theassay according to methods well known in the art. As indicated above, awide variety of labels may be used, with the choice of a label dependingon the sensitivity required, ease of conjugation of the compound,stability requirements, available instrumentation, and disposalprovisions.

[0112] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (for example, biotin) is covalently boundto the molecule. The ligand then binds to an anti-ligand (for example,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. A number ofligands and anti-ligands can be used. Where a ligand has a naturalanti-ligand, for example, biotin, thyroxine, and cortisol, it can beused in conjunction with the labeled, naturally occurring anti-ligands.Alternatively, any haptenic or antigenic compound can be used incombination with an antibody.

[0113] The molecules can also be conjugated directly to signalgenerating compounds, for example, by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, for example, luminol.

[0114] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence, for example, bymicroscopy, visual inspection, via photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels are detectedby providing appropriate substrates for the enzyme and detecting theresulting reaction product. Finally, simple calorimetric labels may bedetected simply by observing the color associated with the label. Thus,in various dipstick assays, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead.

[0115] The ligands of the invention can also be used to detect targetsextracted into solution from a solid material. For example, a solidsample can be extracted with an aqueous, an organic solvent or acritical fluid and the resultant supernatant can be contacted with theligand. Examples of solid samples include animal-derived products,particularly those that have been exposed to agents that transmitprions, for example, bone meal derived from bovine sources. Ligands insome embodiments can be used to detect the presence of prion protein insoil. Other solid samples include brain tissue, corneal tissue, fecalmatter, bone meal, beef by-products, sheep, sheep by-products, deer andelk, deer and elk by-products, and other animals and animal derivedproducts.

[0116] Alternatively, the prion-ligand complexes may be treated with PK.PrPc is highly sensitive to PK, while PrPsc is partially digested toform PrPres. The PrPres molecule itself is highly resistant toproteolysis. Thus, PK treatment will digest PrPc, and will convert PKsensitive PrPsc to PrPres. Following removal of PK, the PrPres can bedenatured and detected by antibodies, such as 3F4.

[0117] In another embodiment, ligands according to the invention may beused for the selective concentration of PrPsc over PrPc.

[0118] Use of Ligands to Quantify Prions

[0119] A ligand-prion complex, or alternatively, an antibody to theligand or ligand-prion complex, can be detected and quantified by any ofa number of means well known to those of skill in the art. These includeanalytic biochemical methods such as spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, and various immunological methods such asfluid or gel precipitation reactions, immunodiffusion (single ordouble), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, and the like.

[0120] Reduction of Non-Specific Binding

[0121] One of skill in the art will appreciate that it is oftendesirable to reduce non-specific binding in assays and during analyteremoval from a sample. Where the assay involves a ligand or othercapture agent immobilized on a solid substrate, it is desirable tominimize the amount of non-specific binding to the solid substrate.Means of reducing such non-specific binding are well known to those ofskill in the art. Typically, this involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine and human serum albumin (BSA), nonfat powdered milk, and gelatinare widely used.

[0122] Other Assay Formats

[0123] Western blot analysis can also be used to detect and quantify thepresence of prion protein in a sample. The technique generally involvesseparating sample products by gel electrophoresis on the basis ofmolecular weight in the presence of SDS, transferring the separatedproteins to a suitable solid support (such as a nitrocellulose filter, anylon filter, or derivatized nylon filter), and incubating the boundsample with the ligands described herein. The ligands specifically bindto a prion peptide fixed on the solid support. These ligands aredirectly labeled or, alternatively, they may be subsequently detectedusing labeled antibodies that specifically bind to the ligand.

[0124] Other assay formats include liposome immunoassays (LIAs), whichuse liposomes designed to bind specific molecules (for example, ligands)and release encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques.

[0125] Pharmaceutical Compositions

[0126] The ligands described herein are useful in therapeutic andprophylactic applications for the treatment of TSEs caused by infectionof a mammal with prion organisms. For instance, in one embodiment, amethod of treating TSEs in a mammal is provided by administering to themammal an effective amount of a pharmaceutical composition containing apharmaceutically acceptable carrier and a synthetic or isolated ligandas described herein. The ligand may prevent polymerization of PrPscthrough inhibition of the binding of PrPsc to PrPsc. In addition it mayprevent inhibit binding of PrPsc to PrPc so decreasing PrPsc mediatedconversion of PrPc to PrPsc thereby delaying the onset of clinicalsymptoms. Moreover, the ligands themselves may be modified by theaddition of a reactive agent to target that molecule to the site ofPrPsc accumulation. Such compositions are suitable for use in a varietyof drug delivery systems.

[0127] Diseases to be treated in accordance with the method include, butare not limited to, BSE, transmissible mink encephalopathy, felinespongiform encephalopathy, CWD, CJD, GSS, fatal insomnia, and vCJD.

[0128] The pharmaceutical compositions are intended for parenteral,topical, oral or local administration. Preferably, the pharmaceuticalcompositions are administered parenterally, for example, intravenously,subcutaneously, intradermally, intranasally or intramuscularly. Thus,the invention provides compositions for administration that comprise asolution of the agents described above dissolved or suspended in anacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be used, for example, water, buffered water, 0.4% saline,0.3% glycine, hyaluronic acid, fibrin sealant and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0129] For solid compositions, conventional nontoxic solid carriers maybe used which include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally approximately 1% to 95%of active ingredient and more preferably at a concentration ofapproximately 25% to 75%.

[0130] For aerosol administration, the polypeptides are preferablysupplied in finely divided form along with a surfactant and propellant.The surfactant must, of course, be nontoxic, and preferably soluble inthe propellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides maybe employed. A carrier can also be included, as desired, as with, forexample, lecithin for intranasal delivery.

[0131] The amount administered will vary depending upon what is beingadministered, the state of the mammal receiving treatment and the mannerof administration. In therapeutic applications, compositions areadministered to a mammal already suffering from prion infection in anamount sufficient to inhibit spread of the prions, or at least partiallyarrest the symptoms of the disease and its complications. An amountadequate to accomplish this is defined as “therapeutically effectivedose.” Amounts effective for this use will depend on the severity of thedisease, the particular composition, and the weight and general state ofthe recipient. Generally, the dose will be in the range of about 1 mg toabout 5 mg per day, preferably about 100 mg per day, for anapproximately 70 kg patient.

[0132] In addition, DNA or RNA encoding the ligands may be introducedinto mammals to obtain the desired therapeutic response to the ligandwhich the nucleic acid encodes.

[0133] The invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are intended neither to limit nor define the invention inany manner.

EXAMPLE 1 Identification of Prion-Binding Ligands

[0134] The prion-binding ligands described in the Tables set forthherein were identified as follows.

[0135] Peptide Library Synthesis

[0136] The peptides and peptide libraries useful for the identificationof the prion-binding ligands described herein were synthesized by eitherPeptides International (Louisville, Ky.) or Commonwealth Biotechnologies(Richmond, Va.) directly on Toyopearl amino resin (TosoBioSep,Montgomeryville, Pa.) using standard Fmoc chemistry based on methodsdescribed by Buettner, et al. 1996. Peptide densities achieved with theabove scheme were typically in the range of 0.1-0.5 mmole/gram resin.Libraries comprising 1, 2, 3, 4, 5 and 6 amino acids were synthesized.The 4, 5 and 6 amino acid libraries were synthesized on amino Toyopearland contained a mixture of tBoc and Fmoc alanine as a spacer between theamino acid and the amino group on the resin. The peptides weresynthesized from the Fmoc alanine and the tBoc was acetylated. Thepresence of “A” was often found in the first position of this library,along with the amino terminal amino acid of the ligand. This wasprobably due to partial deacylation during peptide synthesis,deprotection and/or Edman degradation during sequencing.

[0137] In some embodiments, individual beads, each carrying multiplecopies of a unique ligand, are immobilized in agarose after previouscontact with a solution containing PrP. Since a large number of ligandscan be synthesized onto the surface of beads, it is possible to produceenormous numbers of beads each of which theoretically bears a uniqueligand. These ligands are screened using the described methods forinitial leads. Once a lead has been identified, additional ligands(sub-libraries) are synthesized based on the lead ligand. Screening ofthese sub-libraries may lead to additional ligands with improvedcharacteristics. Through a process of iteration of synthesis andscreening it is possible to identify preferred ligands.

[0138] Peptide Library Binding Screening

[0139] Varying amounts of beads (5-500 mg of dry beads) from a librarywere placed into a Bio-Spin® disposable chromatography column (Bio-RadLaboratories, Cat.# 732-6008), and were washed with 20 column volumes(CV) of 20% MeOH in H₂O to remove possible impurities and organicsolvents used in peptide synthesis. The beads were then washed andequilibrated using 20 CV of 1×TBS, pH 7.6 (1×TBS was prepared by 10-folddilution of 10×TBS, BioSource International, Camarillo, Calif. Cat. #616US-000). The flow was then stopped and the beads were suspended in 1ml of fresh 1×TBS and allowed to swell for an additional 15 minutes. TBSwas drained by gravity and the column was closed. To preventnon-specific binding of test material to the resin 1 mL of Blocker™Casein in TBS (Pierce, Rockford, Ill. Cat # 37532) solution with added0.5% BSA (Sigma, Cat# A-7030) was applied to the beads. After coveringboth ends of the column, blocking was performed overnight at 4° C.,under gentle agitation. The blocking solution was drained, and 1 ml oftest material containing PrPr, PrPc and/or PrPsc was added to the resin.The column was tightly closed at both ends placed in horizontalposition, and gently agitated at room temperature, for three hours. ThePrP-containing material was drained out and beads were washed undergravity, driven with 10 mL of TBS containing 0.05% Tween 20 followed by10 mL of TBS.

[0140] Detection of Bound PrPc

[0141] Detection of normal PrPc was performed using mouse monoclonalantibody 3F4 (Signet, Dedham, Mass.) diluted 1:8,000 in TBS containing1% casein. The monoclonal antibody binds haPrPc, huPrPc and huPrPr, buthas extremely little, or no affinity for haPrPsc or huPrPsc; however, itdoes bind denatured haPrPsc and huPrPsc. One milliliter of diluted 3F4antibody was added to beads from a combinatorial library previouslyexposed to material containing PrPc. The beads were gently agitated with3F4 at room temperature, for one hour. Solution containing non-boundantibody was drained out and the beads were washed with 10 mL of TBS and10 mL of TBS containing 0.1% Tween 20. The beads were then incubated in1 mL of alkaline phosphatase labeled Goat Anti-Mouse IgG (γ) (KPL,Gaithersburg, Md. Cat #741806.) diluted 1:2,000 in 0.5% casein/0.5% BSAin TBS. Incubation was carried out with gentle agitation for 1 hour atroom temperature. Solution containing non-bound secondary antibody wasdrained out and the beads were washed with 10 mL of TBS and 10 mL ofT-TBS. Next, 1 mL of ImmunoPure Fast solution, a substrate for alkalinephosphatase (Pierce, Rockford, Ill., cat. #34034) was prepared asdescribed by the manufacturer and applied to the beads. Incubationproceeded at room temperature for about 15 minutes or until beadsstarted turning light pink, and few dark red beads appeared. Thesubstrate solution was drained and the beads washed with 10 mL of TBS.

[0142] Detection of PrP-Binding Beads Embedded in Agarose

[0143] Identification of PrP-binding beads embedded in agarose wasperformed as follows. First, the base layer of agarose was prepared bycovering the surface of a 49 cm² tray with 9 ml of 1% agarose (LifeTechnologies, Grand Island, Nebr., cat. #15510-027) dissolved in water,which was previously melted and cooled to about 60° C. The agarose wasallowed to solidify. Beads were contacted with test material containingprion protein and washed in TBS as described above. Next, theconcentration of beads was adjusted according to the desiredconcentration of the beads in the gel. A good spread of the beads wasfound at 1.9 mg dry weight equivalent/ml. 90 μl of bead slurry was addedto 800 μl of 0.5% low melting point agarose (BioWhittaker, Rockland, Me.cat. #50111) that had been dissolved in water, melted and cooled toabout 40° C. The mixture was gently vortexed very briefly and pouredover the surface of the base layer. An aliquot of PrP containingmaterial was placed directly into the gel at its corner and served as apositive control for the next procedures. The gel was allowed tosolidify at 4° C.

[0144] Chemiluminescent Detection of PrP-Binding Beads Embedded inAgarose

[0145] After embedding the beads in the gel, a solution of CDP-Star(Applied Biosystems, Bedford, Mass. cat. #MS100R) was added to cover thesurface of gels which were then incubated for 5 minutes as described inthe manufacturer's instructions protocol. Gels were drained of surplussubstrate solution, then placed on a transparency, sealed in a plasticbag and exposed to autoradiography film for 30 minutes. The filmsidentified the location of PrPc or PrPr by spots aligning with red beadsin the gel. These films were subsequently used to align films additionalfilms obtained after denaturing transfer of proteins to a nitrocellulosemembrane.

[0146] Protocol for Protein Transfer from the Embedded Beads toNitrocellulose Membrane.

[0147] This transfer methodology elutes proteins from beads andtransfers them through capillary action onto nitrocellulose or PVDFmembrane. A piece of 3MM filter paper acts to wick transfer buffer(which can be any buffer that is suited to the particular needs of theexperiment) from a tank through the gel. The 3MM wick is pre-wetted withtransfer buffer and placed on a surface with the ends of the paperimmersed in the buffer tank. The gel is placed, soft agar side up, onthe wet 3MM, making sure that there are no bubbles between the paper andthe gel. A piece of membrane (ECL-standard nitrocellulose HybondAmersham, Germany, cat. # RPN303D) cut to the size of the gel is wettedin the transfer buffer and placed on top of the gel. A pipette is rolledover the membrane to eliminate bubbles. Two pieces of pre-wetted 3MMpaper are then placed on the membrane and rolled with a pipette toremove air bubbles. A stack of dry paper towels or other absorbent paperis placed on top, and weighted with 300 g weight. Transfer can proceedas long as necessary.

[0148] Protocol for Chemiluminescence (ECL) Detection

[0149] The membranes are removed from the top of the gels, rinsed, andplaced in plastic containers with 10 mL of 5% (w/v) dried, fat-free milkGiant Fod Inc., Landover, Md. in TBS plus Tween (T-TBS). The membranesare incubated with the milk with gentle agitation for up to 16 hours at4° C., or two hours at room temperature, to prevent non-specific bindingof antibodies to the membranes. After blocking with milk, the membranesare incubated with 10 ml of a 1:8,000 dilution of primary antibody, 3F4,in 5% milk in TBS plus Tween (T-TBS). Incubation is allowed to continuewith gentle agitation for 1.5 hours at room temperature (20-25° C.). Theprimary antibody solution is then discarded and the membranes rinsedtwice with T-TBS, then washed for 15 minutes in T-TBS, then twice forfive minutes in fresh T-TBS. All washes are performed with gentleagitation. Each membrane is then incubated for 1.5 hours at roomtemperature with gentle agitation with 10 ml of a 1:10,000 dilution ofhorse radish peroxidase (HRP) labeled secondary antibody (KPL,Gaithersburg, Md.) in 5% milk in T-TBS. The secondary antibody solutionis then discarded and the membranes rinsed and washed as above. Someexperiments used alkaline phosphatase labeled secondary antibody fordetection of primary antibody.

[0150] Chemiluminescent detection is accomplished by preparing“Chemiluminescent Substrate” (Supersignal, Pierce Rockford Ill. cat#34080) according to the manufacturer's instructions. Ten milliliters ofthe mixture is added to each membrane, protein side up. The substrate isgently swirled manually for five minutes, and the substrate-saturatedmembranes removed and placed on 3MM filter paper to drain quickly, thenwrapped in Sheet Protector (Boise Cascade Office Products, #L2A9113-NG).The protein side of the membranes is exposed to autoradiography film forvarious periods of time and the films developed.

[0151] Detection of Trimer-Binders Specific for PrPsc from ScrapieHamster Brain

[0152] Different biochemical properties between PrPc and PrPsc and thebinding of antibodies, that is, 3F4, were exploited to screen forligands that selectively bind to PrPsc. The monoclonal antibody 3F4binds to denatured PrPsc with considerably higher affinity than tonon-denatured PrPsc. (Safir, J. et al. Eight Prion Strains Have PrPscMolecules With Different Conformations. 1998. Nature Medicine4:1157-1165)

[0153] Ten percent (w/v) homogenates of uninfected and scrapie-infectedhamster brains were prepared in PBS and stored frozen at −80° C.(courtesy of Dr. Robert Rohwer, Va. Medical Center, Baltimore). Prior touse they were thawed on wet ice, and 1.2 ml (uninfected) and 0.5 ml(infected) homogenates, were solubilized in the presence of sarcosyl ata final concentration of 0.5% (w/v) sarcosyl with gentle agitation for30 minutes at room temperature. The samples were centrifuged at 14,000rpm for five minutes, and the supernatants containing, PrPc (uninfected)and a mixture of PrPc and PrPsc (infected), were collected. PrPsc isover represented in scrapie-infected hamster brain tissue relative toPrPc. Five milliliters of brain material for analysis was prepared bycombining 1 ml of normal hamster 10% brain homogenate in 0.5% sarcosylwith 0.33 ml of scrapie-infected brain material and 3.67 ml of TBSbuffer (Pierce, Rockford, Ill.) containing 1% of casein and 1% of BSA(Sigma, St. Louis, Mo.). The final ratio of normal to scrapie-infectedbrain homogenate was 3:1 which gave very approximately equivalentamounts of PrPc and PrPsc. This material was contacted with the trimerbead library and processed according to the procedures. Followingwashing, the beads were variously treated. In one method, they wereincubated with PK to digest PrPc bound to the beads, in another, theywere stained for the presence of PrPc. This was accomplished byincubation of the beads with 3F4 antibody, washing, then addingphosphatase conjugated secondary antibody specific for 3F4, washing andadding a phosphatase substrate, to visualize beads binding PrPc, 3F4,secondary antibody or phosphatase. Thus, those beads that bound PrPcwere red. Once embedded in the gel a second chemiluminescent substratespecific for phosphatase was added, in some experiments, to produce alight signal from the red beads. PrPc, PrPsc and PrPres were transferredfrom the agarose as described above in the presence of 6 Mguanidinium/HCl, which also caused the denaturation of the prionprotein. Denaturation and immobilization of PrPsc on the capturemembrane facilitated the immunodetection of PrPsc, as well as PrPc. Uponalignment of these spots with the previously stained beads, differentpopulations of beads are possible. Those beads that directly bounddetection reagents such as 3F4 and those that bound PrPc plus PrPsc, orPrPc alone would be stained red. Those beads that bound only orpreferentially PrPsc would produce a signal on the membrane, but shouldnot be stained red. These were selected as PrPsc specific beads thoughthey were further tested as beads that might theoretically bind bothPrPc and/or PrPsc at a site on the prion protein that prevented bindingof 3F4. In FIG. 1, beads from the trimer library that did not producedthe signal at the first chemiluminescent detection (before denaturingstep), but produced the signal at the second chemiluminescent detection(after denaturing step), and therefore, were candidates for sequencing,were assigned with numbers.

[0154] Various versions of the methodology described in this Example aregiven in the Tables set forth herein.

[0155] For example, in Tables 10A and B, below, screening of 6-merlibraries (100-300 μm and 65 μm) was performed in presence of sporadicCJD brain material spiked into normal human plasma. Beads were exposedto 0.5% brain homogenate spiked into normal human plasma collected intoCPD, and then were treated with PK 100 μg/ml. To confirm that PK doesnot fully digest peptides from the beads, the resins were treated with1% (w/v) casein and 5% (w/v) human serum albumin and 100 μg/ml of PKprior to sequencing. TABLE 10A Six-amino acid sequences that bind tohuPrPc, huPrPsc or both Screened Bead Light production SEQ IN NOmaterial Sequence color after denaturation 154 huPrPsc RES(na)NVA WhiteStrong no PK* 155 * ES(na)PRQA White Strong 156 * VARENIA White Strong157 * RWEREDA Pink Strong 158 HuPrPsc EWWETV White Medium no PK** 159 **SVYQLDA White Medium 160 ** (na)HEFYGA White Medium 161 ** HE(na)(na)LVAWhite Medium 162 ** A(na)VPV(na)A Pink Medium 163 ** YFDYWLA Pink Medium164 ** FE(na)HRQA Pink Medium 165 ** WRHEPAA Red Medium 166 huPrPsc +SS(na)KKDA White Medium PK*** 167 *** R(na)DKEAA White Medium 168huPrPsc + (na)HEIFPA NA Medium PK**** 169 **** KWYHHRA NA Medium 170**** HWWPHNA NA Medium 171 **** HWQVFYA NA Medium 172 **** FHE(na)EIA NAMedium 173 **** HADF(na)QA NA Medium

[0156] TABLE 10B Six-amino acid sequences that bind to huPrPsc ScreenedBead Light signal after SEQ ID NO material Sequence color denaturation174 HuPrPsc + ALHFETA White Weak PK* 175 * DDPTGFA White Weak 176 *VAPGLGA White 177 * IFRLIEA White Weak 178 * GLERPEA White Weak 179 *IVVRLWA Pink Weak 180 * WHNPHYA Pink Weak 181 * LIYKSDA Pink Weak 182huPrPsc EKPIFNA White Weak no PK** 183 ** HWSEPAA Red Weak 184 **GHNWKEA Pink Strong 185 ** YWHHDDA Pink Strong 186 ** GYPKENA PinkStrong 187 ** PVYWLYA White Strong 188 huPrPsc FGEHTPA White Weak noPK*** 189 *** FQGTREA White Strong 190 *** TGTNRYA White Strong 191 ***KWATRYA White Strong 192 *** NSTKFDA Pink Strong 193 *** LIYKEEA PinkStrong 194 *** EHATYRA White Strong 215 (Control) **** DRDLTFA WhiteNone 216 (Control) **** HNWWIIA White None 217 (Control) **** EVKIGNAWhite None

[0157] In Table 10C, below, screening of 3-mer library was performed inthe presence of brain homogenate prepared from a patient with sporadicCJD (huPrPsc) and beads were treated with PK before the immunodetectionof PrP specific binders. In this assay, 10 mg of resin per column wasincubated with 1 ml of 0.5% (w/v) or 1% (w/v) brain homogenate dilutedinto CPD and containing 0.05% or 0.1% (v/v) sarcosyl, respectively, and0.2 mM of the protease inhibitor (PMSF). Appropriate washes described inthe general protocol were performed, and beads were treated with 1 ml ofPK (100 μg/ml) at 37° C. for one hour. Then followed the generalprocedures described above. Sequences obtained in two experiments arelisted in Table 1° C. The appropriate concentration of brain homogenatematerial present during the incubation is indicated for each group ofsequences.

[0158] In Table 10D, resin from a combinatorial library in the amount of5 mg of dry weight per column was incubated with 1 ml of 0.1% (w/v)brain homogenate diluted into PBS or CPD and containing 0.01% (v/v)sarcosyl and 0.2 mM of PMSF. All the procedures were performed accordingto above mentioned protocols. TABLE 10C Three-amino acid sequences thatbind to huPrPc, huPrPsc, or both Material Light signal after SEQ ID NOscreened Sequence Bead color denaturation 195 huPrPsc + HND White MediumPK* 196 * HER Red Medium 197 * HGD Red Strong 198 * HSD Red Strong 199 *HFD Red Strong 200 **** WND Red None 201 **** YEH Red None 202 **** HWDRed None 203 **** YHD Red None 204 **** YDW Red None 205 **** WDY RedNone 218 (Control) ***** SIV White None 219 (Control) ***** AYP WhiteNone 206 huPrPsc HYD (3x) Red Strong no PK** 207 ** HWD Red Strong 208** WTD Red Strong 209 huPrPsc + FPK White Medium PK*** 210 *** HWK WhiteMedium 211 *** WEE White Medium 212 *** LLR White Medium

[0159] TABLE 10D Three-amino acid sequences that bind to huPrPc,huPrPsc, or both Material Light signal after SEQ ID NO screened SequenceBead color denaturation 147 huPrPsc in WEH Red Strong CPD* 152 * YDY RedStrong 206 * HYD Red Strong 213 huPrPsc in SYF White Weak PBS** 214 **EYY Red Strong

EXAMPLE 2 Secondary Screening of Ligands

[0160] The following examples provide-information on secondary screeningof ligands selected from the various libraries during the primaryscreening to further confirm that the ligands bind PrP.

[0161] Binding of PrPc from normal human brain to trimer resins is shownin FIG. 2. Ten mg of each resin (Amino, HYD (SEQ ID NO:206)), RWD (SEQID NO:113), SYA (SEQ ID NO:108), SYF (SEQ ID NO:213), and YEY (SEQ IDNO:154)), per column was used. The amino resin is the base polymer fromwhich the peptides are synthesized and has some affinity to prionprotein. Resins were equilibrated with either PBS, or CPD at pH 7.4.Frozen normal human brain tissue was used as the source of huPrPc. Itwas first thawed on wet ice. A sample of 10% brain homogenate preparedin PBS or in CPD was solubilized with 1% Sarcosyl and clarified bycentrifugation at 14,000 rpm for five minutes. The supernatant wasrecovered and diluted 100 times to a final concentration of brainhomogenate and Sarcosyl of 0.1% and 0.01%, respectively. One milliliterof this material was applied to the column and the flow through wascollected. Beads were washed with 20 ml of PBS or CPD, and 1 mg of beads(dry weight) was used for evaluation of PrPc binding by Western blot asdescribed below.

[0162] After washing, approximately 1 mg dry weight equivalent of beadswas suspended in 100 ul of buffer, and was heated at 100° C. for 10minutes in 30 μl of Laemmli buffer containing 2% β-mercaptoethanol. Thebeads were centrifuged at 14,000 rpm for one minute, and the supernatantwas evaluated by Western blotting and probing for PrP. Samples wereresolved on NuPAGE 12% Bis-Tris gel (Invitrogen Life Technologies,Carlsbad, Calif., USA) under reduced denaturing conditions, andelectroblotted to nitrocellulose membrane (Invitrogen Life Technologies,Carlsbad, Calif., USA). Specific PrP bands were visualized usingmonoclonal antibody 3F4 diluted 1:10,000. The blots were developed usingSuperSignal West Pico detection system (Pierce, Rockford, Ill., USA)containing chemiluminescent reagent for horseradish peroxidasedetection. Signals were recorded on X-Omat™ Blue XB-1 film (EastmanKodak Company, Rochester, N.Y., (SEW ID NO:113), SYA (SEQ ID NO:108),WEY (SEQ ID NO:102), WSD (SEQ ID NO:115), YID (SEQ ID NO:112), YFE (SEQID NO:106), YEY (SEQ ID NO:154), and WQD) per column was used andprocessed according to the general protocol described above. Columnswere equilibrated with PBS, pH 7.4 Frozen brain tissue from a sporadicCJD patient was used for the PrPsc preparation. It also contained PrPc.Sample of 10% brain homogenate was prepared in PBS treated with 1%sarcosyl and clarified by centrifugation at 14,000 rpm for five minutes.The supernatant was recovered and diluted 100 times to give a finalconcentration of brain homogenate and Sarcosyl of 0.1% and 0.01%,respectively. One milliliter of this material was applied to the beadsand incubated at room temperature in a batch format for three hours. Thebeads were then washed with 20 ml of PBS, and 1 mg of beads (dry weight)was incubated with PBS or with PK (100 μg/ml) in PBS at 37° C. for onehour. These conditions fully digested PrPc. Thus, this helped todiscriminate between PrPsc and PrPc specific-ligands. The usualprocessing of the beads for Western blot followed as described inExample 1.

[0163] Binding of PrPsc in a brain homogenate taken from a sporadic CJDpatient to resins in a flow-through format is shown in FIG. 4. Fiftymilligrams of each resin (Amino, RWERED (SEQ ID NO:157), LW (SEQ IDNO:50), EYY (SEQ ID NO:214), HYD (SEQ ID NO:206)), RWD (SEQ ID NO:113),SYA (SEQ ID NO:108), SYF (SEQ ID NO:213), and YEY (SEQ ID NO:154)) wasused in experiment. The Captiva 96-well Filter Plate (CaptiVac VacuumSistem, ANSYS Technologies, Inc, Cat.# 796) was used instead ofindividual columns. Resins were prepared according to the generalprotocol described above. Resins were equilibrated with CPD at pH 7.4.Frozen brain tissue from a sporadic CJD patient was used as the sourceof huPrPc and huPrPsc. A sample of 10% brain homogenate was prepared inCPD treated with 1% sarcosyl and clarified by centrifugation at 14,000rpm for five minutes. The supernatant was recovered and diluted tentimes to give a final concentration of brain homogenate and sarcosyl of1% and 0.1% respectively. To each well, 250 μl of this material wasapplied. The material was allowed to pass through the resin undergravity with a contact time of about four minutes and flow through wascollected. Resins were washed with 2.5 ml of CPD. One milligram of beads(dry weight) was incubated with PK (100 μg/ml) at 37° C. for one hour.The usual processing of the beads for Western blot followed, asdescribed above.

[0164] Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and material are described above. Allpublications, patent applications, patents and other cited referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

[0165] The foregoing description is provided for describing variousembodiments relating to the invention. Various modifications, additionsand deletions may be made to these embodiments and/or structures withoutdeparting from the scope and spirit of the invention.

EXAMPLE 3 Visualization of PrPc Bound to Resins

[0166] To visualize the binding of PrPc to affinity resins, normal brainhomogenate was bound to amino DVR (SEQ ID NO:114) resin in a columnformat, and the location of the protein in the interior and at theexterior of the beads was visualized by a chromogenic substrate. A 0.5ml column of affinity ligand DVR (SEQ ID NO:114), which was synthesizedon Toyopearl 650-M amino resin, was packed into a PIKSI column (ProMeticBioSciences Ltd, Montreal, Quebec, Canada). To the column was applied1.5 ml of 1% normal hamster brain homogenate (HaBH) diluted in a workingbuffer (WB) (20 mM citrate, 140 mM NaCl, pH 7.0) at a flow rate of 0.5ml/min, which was controlled by a peristaltic pump. Following loading ofHaBH, the columns were washed with 5 ml of WB. The beads were removedfrom the column, chopped with a razor blade to expose the interior ofthe beads, and incubated with primary antibody 3F4 diluted 1:4000 in 1%casein buffer (Pierce, Rockford, Ill.) for 1 hr at room temperature withagitation through rotating. The beads were washed with TBS, pH 7.4(Invitrogen Life Technologies, Carlsbad, Calif., USA) and incubated for1 hr at room temperature with rotation with an alkaline-phosphataselabeled secondary goat anti-mouse antibody (KPL, Gaithersburg, Md.)diluted 1:1000 in 1% casein. The beads were washed with 10 ml TBS at pH7.4, followed by 5 ml TBS at pH 9.5. The beads were incubated withBCIP/NBT alkaline phosphatase substrate (Sigma-Aldrich, St. Louis, Mo.)for several hours and observed under a stereomicroscope. The exteriorsurface of the beads was stained brown/blue, but the interior surfaceremained white, indicating that the protein bound to the exterior of thebeads.

EXAMPLE 4 Removal of PrPsc from Red Blood Cell Concentrates

[0167] Red Blood Cell Concentrates (RBCCs) were spiked with brainhomogenate from hamsters infected with Scrapie at concentrations ordersof magnitude higher than that likely to be found endogenously in theblood of infected animals. The spiked RBCCs were passed in successionthrough columns of resins with various affinity ligands in order toevaluate the ability of the affinity ligands to bind and remove PrP,when present at high concentration, from RBCCs.

[0168] Ten units of type O negative red blood cell concentrates (RBCCs)were leukoreduced on Pall Leukotrap filters (Pall, East Hills, N.Y.),pooled, and spiked with 0.1% scrapie hamster brain homogenate in 0.1%sarkosyl. The spike was added at 2 ml/min, with agitation. The spikedRBCCs were subdivided into 10 equal portions of 300 ml each intotransfer bags (Fenwal Products, Baxter Healthcare Corporation, Deerfild,Ill.).

[0169] Five columns, each containing 10 ml of a specific resin, were setup in series, so that the flow through of column one, containing unboundmaterial was applied to column two. This was continued until all 5columns were exposed to RBCCs. Through column one, 300 ml of spikedRBCCs was passed, the flow through collected, and run over column two,and so on, until all of the columns were exposed to RBCCs. The beads inthe column were collected, and 100 μl sample of beads was washed, anddivided into two portions. One portion was incubated with Proteinase K(in Table 11, sample incubated with Proteinase K is denoted +PK, samplenot incubated with Proteinase K is denoted −PK) at 1 mg/ml for 1 hr at37° C. The proteins that bound to both the +PK and −PK beads were elutedfrom the beads by boiling in 2× sample buffer (NuPAGE, HelixxTechnologies Inc., Toronto, Ontario, Canada). Each sample in the amountof 10 μl of was loaded on a 12% Bis-Tris SDS-PAGE gel (Invitrogen) andelectrophoresed for 45 min. The proteins from the gel were transferredto a membrane and the PrP protein was detected in a Western blot usingmouse anti-human PrP antibody 3F4 as the primary antibody, goatanti-mouse alkaline phosphatase conjugated antibody as the secondaryantibody, and detected with Western Breeze chemiluminescent detection(Invitrogen). The bands on the gel, obtained by eluting resin-boundprotein, indicate the presence of PrPres on the beads that were derivedfrom the flow-through of the previous column (or starting material inthe case of column 1).

[0170] PrPres was found on beads from columns 1-5 for the negativecontrol, acetylated SYA (Ac-SYA) (SEQ ID NO:108) resin, indicating thatthis resin did not bind PrPsc. PrPres was found in high amounts oncolumn 1, and in decreased amounts on column 2 for DVR (SEQ ID NO:114)and SYA (SEQ ID NO:10), with only a small amount of PrPres present onbeads from column 3. This indicated that these resins remove all of thePrPres to the limit of detection of the Western blot in 3 columns, or 30ml of resin. Resins YVHEA (SEQ ID NO:63) and (D)ES(na)PRQ-EACA (SEQ IDNO:226-EACA) also show decreasing amounts of PrPres on columns 1 through3; however, there is more PrPres bound to column 3 than in the previoustwo resins. An equivalent amount of PrPres is found on every column ofWFVEA (SEQ ID NO:225), indicating that this resin binds a small amountof PrPres on every column, but does not bind and remove all of the prionprotein to the limit of detection. As the spike was several fold higherthan the amount of PrP that has been observed endogenously in the bloodof animals, these results indicated that certain of these resins had theability to remove most, if not all of the endogenous PrPres present inblood. TABLE 11 Gel loading pattern for electrophoresis of samples inExample 4. Resins DVR (SEQ ID NO: 114), YVHEA (SEQ ID NO: 63), ResinsSYA & Ac-SYA (D)ES(na)PRQ (SEQ ID NO: 226), (SEQ ID NO: 10) WFDEA (SEQID NO: 225)  1. MWM  1. MWM  2. Column #1 − PK  2. Column #1 − PK  3.Column #1 + PK  3. Column #1 + PK  4. Column #2 − PK  4. Column #2 − PK 5. Column #2 + PK  5. Column #2 + PK  6. Column #3 − PK  6. Column #3 −PK  7. Column #3 + PK  7. Column #3 + PK  8. Column #4 − PK  8. Column#4 − PK  9. Column #4 + PK  9. Column #4 + PK 10. Scrapie brainhomogenate − PK 10. Column #5 − PK    (1:100) 11. Scrapie brainhomogenate − PK 11. Column #5 + PK    (1:10) 12. Scrapie brainhomogenate − PK 12. Scrapie brain homogenate −    (1:2) PK (1:100) 13.Scrapie brain test material − PK 13. Scrapie brain homogenate −    PK(1:10) 14. Scrapie brain test material + PK 14. Scrapie brain homogenate−    PK (1:2) 15. MWM 15. Scrapie brain test material −    PK 16.Scrapie brain test material +    PK 17. MWM

[0171]

1 226 1 5 PRT prion sp. 1 Arg Tyr Pro Gly Gln 1 5 2 6 PRT prion sp. 2Asp Arg Tyr Tyr Arg Asp 1 5 3 6 PRT prion sp. 3 Gln Ala Tyr Tyr Gln Arg1 5 4 6 PRT prion sp. 4 Gln Val Tyr Tyr Arg Pro 1 5 5 7 PRT ArtificialSequence prion-binding ligand 5 Lys Ile His Lys Phe Leu Ala 1 5 6 7 PRTArtificial Sequence prion-binding ligand 6 Gly Thr His Asp Phe Gln Ala 15 7 7 PRT Artificial Sequence prion-binding ligand 7 Lys Phe Gly Ser ThrHis Ala 1 5 8 7 PRT Artificial Sequence prion-binding ligand 8 Phe ValAsn Glu Ile Glu Ala 1 5 9 7 PRT Artificial Sequence prion-binding ligand9 Gly Leu His Phe Lys Ser Ala 1 5 10 7 PRT Artificial Sequenceprion-binding ligand 10 Gly Arg Val Leu His His Ala 1 5 11 7 PRTArtificial Sequence prion-binding ligand 11 Gln Lys Asn Ser Glu Trp Ala1 5 12 7 PRT Artificial Sequence prion-binding ligand 12 His Ala Tyr PheThr His Ala 1 5 13 7 PRT Artificial Sequence prion-binding ligand 13 TrpPro Lys Gly Ala Val Ala 1 5 14 7 PRT Artificial Sequence prion-bindingligand 14 Arg Pro Trp Lys Lys Ala Ala 1 5 15 7 PRT Artificial Sequenceprion-binding ligand 15 Pro Lys His Ile Trp Pro Ala 1 5 16 7 PRTArtificial Sequence prion-binding ligand 16 His Lys Leu Trp Gly Val Ala1 5 17 7 PRT Artificial Sequence prion-binding ligand 17 Gly Gly Tyr LysPro Tyr Ala 1 5 18 7 PRT Artificial Sequence prion-binding ligand 18 GluAsn Val Ser Gln Asn Ala 1 5 19 7 PRT Artificial Sequence prion-bindingligand 19 His Thr Tyr Tyr Asn Gly Ala 1 5 20 7 PRT Artificial Sequenceprion-binding ligand 20 Lys Lys Lys Ser Asp His Ala 1 5 21 7 PRTArtificial Sequence prion-binding ligand 21 His His Leu Lys Gly Thr Ala1 5 22 7 PRT Artificial Sequence prion-binding ligand 22 Lys Lys His GlyVal Trp Ala 1 5 23 7 PRT Artificial Sequence prion-binding ligand 23 AspGly Thr Gln Ala His Ala 1 5 24 7 PRT Artificial Sequence prion-bindingligand 24 Ala Pro His Arg Asn Asn Ala 1 5 25 7 PRT Artificial Sequenceprion-binding ligand 25 His His Gly His Asn Ile Ala 1 5 26 7 PRTArtificial Sequence prion-binding ligand 26 His Thr Trp His Gly Gln Ala1 5 27 7 PRT Artificial Sequence prion-binding ligand 27 His Val Phe ValThr Trp Ala 1 5 28 7 PRT Artificial Sequence prion-binding ligand 28 ThrHis His Phe Tyr Ile Ala 1 5 29 7 PRT Artificial Sequence prion-bindingligand 29 Lys Leu Gly Trp Gly Xaa Ala 1 5 30 7 PRT Artificial Sequenceprion-binding ligand 30 Gly Ser Lys Lys Lys Glu Ala 1 5 31 7 PRTArtificial Sequence prion-binding ligand 31 Pro Leu Leu Val Val Trp Ala1 5 32 7 PRT Artificial Sequence prion-binding ligand 32 Trp Leu Leu ValGly Gly Ala 1 5 33 7 PRT Artificial Sequence prion-binding ligand 33 XaaGln Val Leu Val Tyr Ala 1 5 34 7 PRT Artificial Sequence prion-bindingligand 34 Arg Arg His Gln Arg Gln Ala 1 5 35 7 PRT Artificial Sequenceprion-binding ligand 35 Leu Pro Trp Thr Phe Gly Ala 1 5 36 7 PRTArtificial Sequence prion-binding ligand 36 Ile Phe Ile Ile Ile Thr Ala1 5 37 7 PRT Artificial Sequence prion-binding ligand 37 Pro Xaa Ile GluPro His Ala 1 5 38 7 PRT Artificial Sequence prion-binding ligand 38 GluTrp Gly Ile Ile Trp Ala 1 5 39 7 PRT Artificial Sequence prion-bindingligand 39 Gly Trp Tyr Ile Tyr Phe Ala 1 5 40 7 PRT Artificial Sequenceprion-binding ligand 40 Thr Leu Ile Leu Phe His Ala 1 5 41 7 PRTArtificial Sequence prion-binding ligand 41 Phe Leu Leu Ser Asn His Ala1 5 42 7 PRT Artificial Sequence prion-binding ligand 42 Trp Gln Ile ArgPhe Phe Ala 1 5 43 7 PRT Artificial Sequence prion-binding ligand 43 ValLeu Leu Val Phe Glu Ala 1 5 44 7 PRT Artificial Sequence prion-bindingligand 44 Gly Trp Val Leu Glu Ile Ala 1 5 45 7 PRT Artificial Sequenceprion-binding ligand 45 Phe Leu Leu Ile Asp Thr Ala 1 5 46 7 PRTArtificial Sequence prion-binding ligand 46 Gly Phe Leu Phe Lys Phe Ala1 5 47 7 PRT Artificial Sequence prion-binding ligand 47 Pro Trp Thr IleTyr Ile Ala 1 5 48 2 PRT Artificial Sequence prion-binding ligand 48 TrpHis 1 49 2 PRT Artificial Sequence prion-binding ligand 49 Trp Trp 1 502 PRT Artificial Sequence prion-binding ligand 50 Leu Trp 1 51 3 PRTArtificial Sequence prion-binding ligand 51 Trp Asn Ala 1 52 3 PRTArtificial Sequence prion-binding ligand 52 Glu Phe Trp 1 53 3 PRTArtificial Sequence prion-binding ligand 53 Leu Pro Trp 1 54 3 PRTArtificial Sequence prion-binding ligand 54 Tyr Glu Tyr 1 55 3 PRTArtificial Sequence prion-binding ligand 55 Trp Pro Ala 1 56 3 PRTArtificial Sequence prion-binding ligand 56 Phe Asn Gln 1 57 3 PRTArtificial Sequence prion-binding ligand 57 Tyr His Glu 1 58 3 PRTArtificial Sequence prion-binding ligand 58 Leu Phe Ala 1 59 3 PRTArtificial Sequence prion-binding ligand 59 Asn His Tyr 1 60 3 PRTArtificial Sequence prion-binding ligand 60 Thr Leu Gly 1 61 3 PRTArtificial Sequence prion-binding ligand 61 Trp Val Asp 1 62 5 PRTArtificial Sequence prion-binding ligand 62 Tyr Trp Asp Gln Ala 1 5 63 5PRT Artificial Sequence prion-binding ligand 63 Tyr Val His Glu Ala 1 564 5 PRT Artificial Sequence prion-binding ligand 64 Trp Phe Asp Glu Ala1 5 65 6 PRT Artificial Sequence prion-binding ligand 65 Leu Gln Trp TyrAsp Ala 1 5 66 6 PRT Artificial Sequence prion-binding ligand 66 Tyr ThrHis Ser Glu Ala 1 5 67 6 PRT Artificial Sequence prion-binding ligand 67Trp Ile Asp Tyr Glu Ala 1 5 68 6 PRT Artificial Sequence prion-bindingligand 68 Val Trp Ile Asp Ala Ala 1 5 69 7 PRT Artificial Sequenceprion-binding ligand 69 Trp Asp Glu Ala Glu Glu Ala 1 5 70 7 PRTArtificial Sequence prion-binding ligand 70 Tyr Asp Ser Tyr Asp Asp Ala1 5 71 7 PRT Artificial Sequence prion-binding ligand 71 Asn Asp Phe IleAsp Phe Ala 1 5 72 7 PRT Artificial Sequence prion-binding ligand 72 TyrGlu Pro Trp Gly Ser Ala 1 5 73 7 PRT Artificial Sequence prion-bindingligand 73 Glu Tyr Gly Asp Trp Trp Ala 1 5 74 7 PRT Artificial Sequenceprion-binding ligand 74 Trp Asp Tyr Asp Gln Glu Ala 1 5 75 7 PRTArtificial Sequence prion-binding ligand 75 Asp Trp Gly Asp Pro Phe Ala1 5 76 7 PRT Artificial Sequence prion-binding ligand 76 Asp Trp Pro GluVal Trp Ala 1 5 77 7 PRT Artificial Sequence prion-binding ligand 77 PheHis Asp Phe Ser Glu Ala 1 5 78 7 PRT Artificial Sequence prion-bindingligand 78 Asp Thr Phe Trp Asp Tyr Ala 1 5 79 7 PRT Artificial Sequenceprion-binding ligand 79 Trp Asn Asp Leu Asp Asn Ala 1 5 80 7 PRTArtificial Sequence prion-binding ligand 80 Ala Ser Ala Leu Val Tyr Ala1 5 81 7 PRT Artificial Sequence prion-binding ligand 81 Leu Ile Asn AlaGly Gly Ala 1 5 82 7 PRT Artificial Sequence prion-binding ligand 82 TrpGlu Ser Tyr Val Thr Ala 1 5 83 7 PRT Artificial Sequence prion-bindingligand 83 Trp Ser Asp Glu Gly Tyr Ala 1 5 84 7 PRT Artificial Sequenceprion-binding ligand 84 Tyr Arg Trp Thr Gly Pro Ala 1 5 85 7 PRTArtificial Sequence prion-binding ligand 85 Tyr Glu Asp Gln Trp Gln Ala1 5 86 7 PRT Artificial Sequence prion-binding ligand 86 Glu Trp Ala AspAsp Asn Ala 1 5 87 7 PRT Artificial Sequence prion-binding ligand 87 TyrGlu Ile Asp Tyr Gly Ala 1 5 88 7 PRT Artificial Sequence prion-bindingligand 88 Glu Phe Gly Tyr Phe Asp Ala 1 5 89 7 PRT Artificial Sequenceprion-binding ligand 89 Trp Gly Asp Glu Gln Asp Ala 1 5 90 7 PRTArtificial Sequence prion-binding ligand 90 His Glu Glu Asp Trp Ala Ala1 5 91 7 PRT Artificial Sequence prion-binding ligand 91 Phe Glu Asp PheGlu Leu Ala 1 5 92 7 PRT Artificial Sequence prion-binding ligand 92 ThrTrp Gly Ile Asp Glu Ala 1 5 93 7 PRT Artificial Sequence prion-bindingligand 93 Trp Asp Pro Thr Asp Tyr Ala 1 5 94 7 PRT Artificial Sequenceprion-binding ligand 94 Asn Asp Lys Ile His Thr Ala 1 5 95 7 PRTArtificial Sequence prion-binding ligand 95 Phe Glu Asp Phe Phe Ser Ala1 5 96 7 PRT Artificial Sequence prion-binding ligand 96 Tyr Glu Trp AlaGlu Gln Ala 1 5 97 7 PRT Artificial Sequence prion-binding ligand 97 ThrHis Val Tyr Phe Leu Ala 1 5 98 6 PRT Artificial Sequence prion-bindingligand 98 Xaa Asp Phe Ser Asp Ala 1 5 99 7 PRT Artificial Sequenceprion-binding ligand 99 Tyr Arg Thr Pro Asn Glu Ala 1 5 100 6 PRTArtificial Sequence prion-binding ligand 100 Xaa Arg Ser Glu Thr Ala 1 5101 3 PRT Artificial Sequence prion-binding ligand 101 Ile His Asn 1 1023 PRT Artificial Sequence prion-binding ligand 102 Trp Glu Tyr 1 103 3PRT Artificial Sequence prion-binding ligand 103 Asp Tyr Trp 1 104 3 PRTArtificial Sequence prion-binding ligand 104 Trp Asp Trp 1 105 3 PRTArtificial Sequence prion-binding ligand 105 Trp Gln Asp 1 106 3 PRTArtificial Sequence prion-binding ligand 106 Tyr Phe Glu 1 107 3 PRTArtificial Sequence prion-binding ligand 107 Asn Tyr Glu 1 108 3 PRTArtificial Sequence prion-binding ligand 108 Ser Tyr Ala 1 109 3 PRTArtificial Sequence prion-binding ligand 109 Trp Asp Leu 1 110 3 PRTArtificial Sequence prion-binding ligand 110 Trp Leu Glu 1 111 3 PRTArtificial Sequence prion-binding ligand 111 Val Gln Arg 1 112 3 PRTArtificial Sequence prion-binding ligand 112 Tyr Ile Asp 1 113 3 PRTArtificial Sequence prion-binding ligand 113 Arg Trp Asp 1 114 3 PRTArtificial Sequence prion-binding ligand 114 Asp Val Arg 1 115 3 PRTArtificial Sequence prion-binding ligand 115 Trp Ser Asp 1 116 3 PRTArtificial Sequence prion-binding ligand 116 His Trp Asp 1 117 3 PRTArtificial Sequence prion-binding ligand 117 Trp Gln Asp 1 118 3 PRTArtificial Sequence prion-binding ligand 118 Trp Asp Asp 1 119 3 PRTArtificial Sequence prion-binding ligand 119 Trp Glu Asp 1 120 3 PRTArtificial Sequence prion-binding ligand 120 Ile Thr Asn 1 121 3 PRTArtificial Sequence prion-binding ligand 121 Tyr Glu Asp 1 122 7 PRTArtificial Sequence prion-binding ligand 122 Arg Val Ala Asp Glu Glu Ala1 5 123 7 PRT Artificial Sequence prion-binding ligand 123 Glu Tyr TyrVal Asp Ala Ala 1 5 124 7 PRT Artificial Sequence prion-binding ligand124 Trp Gln Asp Phe Asn Leu Ala 1 5 125 7 PRT Artificial Sequenceprion-binding ligand 125 Tyr Asp Asn Pro Ile Asp Ala 1 5 126 7 PRTArtificial Sequence prion-binding ligand 126 Tyr Phe Asn Glu His Glu Ala1 5 127 7 PRT Artificial Sequence prion-binding ligand 127 Glu Trp GlyAla Asp Gly Ala 1 5 128 7 PRT Artificial Sequence prion-binding ligand128 Asp Val Ile Tyr Ser His Ala 1 5 129 7 PRT Artificial Sequenceprion-binding ligand 129 Trp His Ile Leu Glu Glu Ala 1 5 130 7 PRTArtificial Sequence prion-binding ligand 130 Asn Pro His Glu Asn Phe Ala1 5 131 7 PRT Artificial Sequence prion-binding ligand 131 His Glu AspAsn Gly Gly Ala 1 5 132 7 PRT Artificial Sequence prion-binding ligand132 Ser Asp Ser Glu Gly Pro Ala 1 5 133 7 PRT Artificial Sequenceprion-binding ligand 133 Glu Phe Gln Glu Phe Thr Ala 1 5 134 7 PRTArtificial Sequence prion-binding ligand 134 Gln Glu Gly Asp Glu Ile Ala1 5 135 7 PRT Artificial Sequence prion-binding ligand 135 Asp Ile TyrAla Glu Thr Ala 1 5 136 7 PRT Artificial Sequence prion-binding ligand136 Asp Arg Val Arg Glu Thr Ala 1 5 137 7 PRT Artificial Sequenceprion-binding ligand 137 Phe Glu Glu Pro Gln Trp Ala 1 5 138 7 PRTArtificial Sequence prion-binding ligand 138 Phe Glu Gly Glu Glu Phe Ala1 5 139 6 PRT Artificial Sequence prion-binding ligand 139 Xaa Phe AsnIle His Ala 1 5 140 3 PRT Artificial Sequence prion-binding ligand 140Tyr Asp Trp 1 141 3 PRT Artificial Sequence prion-binding ligand 141 AsnTyr Thr 1 142 3 PRT Artificial Sequence prion-binding ligand 142 Ser TyrThr 1 143 3 PRT Artificial Sequence prion-binding ligand 143 Trp Ala Asp1 144 3 PRT Artificial Sequence prion-binding ligand 144 Gln Trp Gly 1145 3 PRT Artificial Sequence prion-binding ligand 145 Trp Gly Asp 1 1463 PRT Artificial Sequence prion-binding ligand 146 Glu Tyr Phe 1 147 3PRT Artificial Sequence prion-binding ligand 147 Trp Glu His 1 148 3 PRTArtificial Sequence prion-binding ligand 148 Leu Tyr Asp 1 149 3 PRTArtificial Sequence prion-binding ligand 149 Asp Tyr Tyr 1 150 3 PRTArtificial Sequence prion-binding ligand 150 Phe Tyr Glu 1 151 3 PRTArtificial Sequence prion-binding ligand 151 Glu Tyr Tyr 1 152 3 PRTArtificial Sequence prion-binding ligand 152 Tyr Asp Tyr 1 153 3 PRTArtificial Sequence prion-binding ligand 153 Trp Asp His 1 154 7 PRTArtificial Sequence prion-binding ligand 154 Arg Glu Ser Xaa Asn Val Ala1 5 155 7 PRT Artificial Sequence prion-binding ligand 155 Glu Ser XaaPro Arg Gln Ala 1 5 156 7 PRT Artificial Sequence prion-binding ligand156 Val Ala Arg Glu Asn Ile Ala 1 5 157 7 PRT Artificial Sequenceprion-binding ligand 157 Arg Trp Glu Arg Glu Asp Ala 1 5 158 6 PRTArtificial Sequence prion-binding ligand 158 Glu Trp Trp Glu Thr Val 1 5159 7 PRT Artificial Sequence prion-binding ligand 159 Ser Val Tyr GlnLeu Asp Ala 1 5 160 7 PRT Artificial Sequence prion-binding ligand 160Xaa His Glu Phe Tyr Gly Ala 1 5 161 7 PRT Artificial Sequenceprion-binding ligand 161 His Glu Xaa Xaa Leu Val Ala 1 5 162 7 PRTArtificial Sequence prion-binding ligand 162 Ala Xaa Val Pro Val Xaa Ala1 5 163 7 PRT Artificial Sequence prion-binding ligand 163 Tyr Phe AspTyr Trp Leu Ala 1 5 164 7 PRT Artificial Sequence prion-binding ligand164 Phe Glu Xaa His Arg Gln Ala 1 5 165 7 PRT Artificial Sequenceprion-binding ligand 165 Trp Arg His Glu Pro Ala Ala 1 5 166 7 PRTArtificial Sequence prion-binding ligand 166 Ser Ser Xaa Lys Lys Asp Ala1 5 167 7 PRT Artificial Sequence prion-binding ligand 167 Arg Xaa AspLys Glu Ala Ala 1 5 168 7 PRT Artificial Sequence prion-binding ligand168 Xaa His Glu Ile Phe Pro Ala 1 5 169 7 PRT Artificial Sequenceprion-binding ligand 169 Lys Trp Tyr His His Arg Ala 1 5 170 7 PRTArtificial Sequence prion-binding ligand 170 His Trp Trp Pro His Asn Ala1 5 171 7 PRT Artificial Sequence prion-binding ligand 171 His Trp GlnVal Phe Tyr Ala 1 5 172 7 PRT Artificial Sequence prion-binding ligand172 Phe His Glu Xaa Glu Ile Ala 1 5 173 7 PRT Artificial Sequenceprion-binding ligand 173 His Ala Asp Phe Xaa Gln Ala 1 5 174 7 PRTArtificial Sequence prion-binding ligand 174 Ala Leu His Phe Glu Thr Ala1 5 175 7 PRT Artificial Sequence prion-binding ligand 175 Asp Asp ProThr Gly Phe Ala 1 5 176 7 PRT Artificial Sequence prion-binding ligand176 Val Ala Pro Gly Leu Gly Ala 1 5 177 7 PRT Artificial Sequenceprion-binding ligand 177 Ile Phe Arg Leu Ile Glu Ala 1 5 178 7 PRTArtificial Sequence prion-binding ligand 178 Gly Leu Glu Arg Pro Glu Ala1 5 179 7 PRT Artificial Sequence prion-binding ligand 179 Ile Val ValArg Leu Trp Ala 1 5 180 7 PRT Artificial Sequence prion-binding ligand180 Trp His Asn Pro His Tyr Ala 1 5 181 7 PRT Artificial Sequenceprion-binding ligand 181 Leu Ile Tyr Lys Ser Asp Ala 1 5 182 7 PRTArtificial Sequence prion-binding ligand 182 Glu Lys Pro Ile Phe Asn Ala1 5 183 7 PRT Artificial Sequence prion-binding ligand 183 His Trp SerGlu Pro Ala Ala 1 5 184 7 PRT Artificial Sequence prion-binding ligand184 Gly His Asn Trp Lys Glu Ala 1 5 185 7 PRT Artificial Sequenceprion-binding ligand 185 Tyr Trp His His Asp Asp Ala 1 5 186 7 PRTArtificial Sequence prion-binding ligand 186 Gly Tyr Pro Lys Glu Asn Ala1 5 187 7 PRT Artificial Sequence prion-binding ligand 187 Pro Val TyrTrp Leu Tyr Ala 1 5 188 7 PRT Artificial Sequence prion-binding ligand188 Phe Gly Glu His Thr Pro Ala 1 5 189 7 PRT Artificial Sequenceprion-binding ligand 189 Phe Gln Gly Thr Arg Glu Ala 1 5 190 7 PRTArtificial Sequence prion-binding ligand 190 Thr Gly Thr Asn Arg Tyr Ala1 5 191 7 PRT Artificial Sequence prion-binding ligand 191 Lys Trp AlaThr Arg Tyr Ala 1 5 192 7 PRT Artificial Sequence prion-binding ligand192 Asn Ser Thr Lys Phe Asp Ala 1 5 193 7 PRT Artificial Sequenceprion-binding ligand 193 Leu Ile Tyr Lys Glu Glu Ala 1 5 194 7 PRTArtificial Sequence prion-binding ligand 194 Glu His Ala Thr Tyr Arg Ala1 5 195 3 PRT Artificial Sequence prion-binding ligand 195 His Asn Asp 1196 3 PRT Artificial Sequence prion-binding ligand 196 His Glu Arg 1 1973 PRT Artificial Sequence prion-binding ligand 197 His Gly Asp 1 198 3PRT Artificial Sequence prion-binding ligand 198 His Ser Asp 1 199 3 PRTArtificial Sequence prion-binding ligand 199 His Phe Asp 1 200 3 PRTArtificial Sequence prion-binding ligand 200 Trp Asn Asp 1 201 3 PRTArtificial Sequence prion-binding ligand 201 Tyr Glu His 1 202 3 PRTArtificial Sequence prion-binding ligand 202 His Trp Asp 1 203 3 PRTArtificial Sequence prion-binding ligand 203 Tyr His Asp 1 204 3 PRTArtificial Sequence prion-binding ligand 204 Tyr Asp Trp 1 205 3 PRTArtificial Sequence prion-binding ligand 205 Trp Asp Tyr 1 206 3 PRTArtificial Sequence prion-binding ligand 206 His Tyr Asp 1 207 3 PRTArtificial Sequence prion-binding ligand 207 His Trp Asp 1 208 3 PRTArtificial Sequence prion-binding ligand 208 Trp Thr Asp 1 209 3 PRTArtificial Sequence prion-binding ligand 209 Phe Pro Lys 1 210 3 PRTArtificial Sequence prion-binding ligand 210 His Trp Lys 1 211 3 PRTArtificial Sequence prion-binding ligand 211 Trp Glu Glu 1 212 3 PRTArtificial Sequence prion-binding ligand 212 Leu Leu Arg 1 213 3 PRTArtificial Sequence prion-binding ligand 213 Ser Tyr Phe 1 214 3 PRTArtificial Sequence prion-binding ligand 214 Glu Tyr Tyr 1 215 7 PRTArtificial Sequence prion-binding ligand 215 Asp Arg Asp Leu Thr Phe Ala1 5 216 7 PRT Artificial Sequence prion-binding ligand 216 His Asn TrpTrp Ile Ile Ala 1 5 217 7 PRT Artificial Sequence prion-binding ligand217 Glu Val Lys Ile Gly Asn Ala 1 5 218 3 PRT Artificial Sequenceprion-binding ligand 218 Ser Ile Val 1 219 3 PRT Artificial Sequenceprion-binding ligand 219 Ala Tyr Pro 1 220 8 PRT Artificial Sequenceprion-binding ligand 220 Pro His Gly Gly Gly Trp Gly Gln 1 5 221 5 PRTprion sp. MISC_FEATURE (4)..(4) Xaa at position 4 is Gly, Pro or Asn 221Arg Tyr Pro Xaa Gln 1 5 222 6 PRT prion sp. MISC_FEATURE (1)..(1) Xaa atposition 1 is any amino acid 222 Xaa Xaa Tyr Tyr Xaa Xaa 1 5 223 4 PRTArtificial Sequence prion-binding ligand 223 Xaa Xaa Xaa Xaa 1 224 4 PRTArtificial Sequence prion-binding ligand 224 Xaa Xaa Xaa Xaa 1 225 5 PRTArtificial Sequence Prion-binding ligand 225 Trp Phe Val Glu Ala 1 5 2267 PRT Artificial Sequence Prion-binding ligand 226 Asp Glu Ser Xaa ProArg Gln 1 5

What is claimed is:
 1. A prion-binding ligand, wherein the ligand iscapable of binding to a peptide having an amino acid sequence RYPxQ (SEQID NO:221), wherein x is G, P or N, or wherein the ligand binds to apeptide having an amino acid sequence xxYYux (SEQ ID NO:222), wherein xis any amino acid and u is R or Q.
 2. The ligand of claim 1, wherein theligand is capable of binding to a peptide having an amino acid sequenceselected from the group consisting of RYPGQ (SEQ ID NO:1), DRYYRD (SEQID NO:2), QAYYQR (SEQ ID NO:3), and QVYYRP (SEQ ID NO:4).
 3. The ligandof claim 1, wherein the ligand has a molecular weight of less thanapproximately 6 kDa.
 4. The ligand of claim 3, wherein the ligand is apeptide having an amino acid sequence of six amino acids.
 5. The ligandof claim 4, wherein the ligand is capable of binding to a peptide havingan amino acid sequence DRYYRD (SEQ ID NO:2), and wherein the ligandamino acid sequence comprises an amino acid lysine (K) or an amino acidhistidine (H).
 6. The ligand of claim 4, wherein the ligand is capableof binding to a peptide having an amino acid sequence QAYYQR (SEQ IDNO:3), wherein the ligand amino acid sequence comprises and amino acidhistidine (H), and wherein the ligand possesses a net positive charge atpH
 7. 7. The ligand of claim 1 wherein the ligand is capable of bindingto a peptide having an amino acid sequence QVYYRP (SEQ ID NO:4), and theligand is a peptide having an amino acid sequence that comprises aminoacid sequences LL, LI, VL, or II.
 8. The ligand of claim 1 wherein theligand is capable of binding to a peptide having an amino acid sequenceQVYYRP (SEQ ID NO:4), and wherein the ligand is a peptide having anamino acid sequence that comprises an aromatic amino acid, and whereinthe ligand is neutrally charged.
 9. The ligand of claim 1, wherein theligand is capable of binding to a peptide having an amino acid sequenceRYPGQ (SEQ ID NO:1), and wherein the ligand is a peptide having an aminoacid sequence selected from the group consisting of SEQ ID NOS:5-13. 10.The ligand of claim 1, wherein the ligand is capable of binding to apeptide having an amino acid sequence DRYYRD (SEQ ID NO:2), and whereinthe ligand is a peptide having an amino acid sequence selected from thegroup consisting of SEQ ID NOS:14-22.
 11. The ligand of claim 1, whereinthe ligand is capable of binding to a peptide having an amino acidsequence QAYYQR (SEQ ID NO: 3), and wherein the ligand is a peptidehaving an amino acid sequence selected from the group consisting of SEQID NOS:23-31.
 12. The ligand of claim 1, wherein the ligand is capableof binding to a peptide having an amino acid sequence QVYYRP (SEQ IDNO:4), and wherein the ligand is a peptide having an amino sequenceselected from the group consisting of SEQ ID NOS:31-47.
 13. Aprion-binding ligand, wherein the ligand is capable of binding to anative form of prion protein (PrPc) and is a peptide having an aminoacid sequence selected from the group consisting of SEQ ID NOS:48-100and SEQ ID NOS:116-139.
 14. The ligand of claim 13, wherein the ligandis capable of binding to a native prion protein that infects humans(huPrPc) and has an amino acid sequence selected from the groupconsisting of SEQ ID NOS:116-139.
 15. A prion-binding ligand, whereinthe ligand is capable of binding to both a native form of prion protein(PrPc) and a conformationally altered form of prion protein (PrPsc) andis a peptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOS:52, 54, 101-115, and 154-173.
 16. The ligand ofclaim 15 wherein the ligand is capable of binding to both a native formof prion protein in humans (huPrPc) and a conformationally altered formof prion protein in humans (huPrPsc) and is a peptide having an aminoacid sequence selected from the group consisting of SEQ ID NOS:154-173.17. A prion-binding ligand, wherein the ligand is capable of binding toa prion protein expressed by recombinant technology (PrPr), and theligand has is a peptide having an amino acid sequence selected from thegroup consisting of SEQ ID NOS:54, 105, and 140-153.
 18. A prion-bindingligand, wherein the ligand is capable of binding to a conformationallyaltered form of prion protein (PrPsc), and the ligand is a peptidehaving an amino acid sequence selected from the group consisting of SEQID NOS:174-194, 147, 152, 206, 213, and
 214. 19. A prion-binding ligand,wherein the ligand is capable of binding to a native form of prionprotein (PrPc) or a conformationally altered form of prion protein(PrPsc) treated with proteinase K, and the ligand is a peptide having anamino acid sequence selected from the group consisting of SEQ IDNOS:195-212.
 20. A method of detecting a prion protein in a sample,comprising: contacting the sample with a ligand capable of binding toone or more prion proteins, a fragment thereof, or a peptide derivedtherefrom under conditions sufficient to cause formation of a complexbetween the prion protein, the fragment thereof, or the peptide derivedtherefrom and the ligand; and detecting the complex in the sample. 21.The method of claim 20 wherein the sample is a biological sample. 22.The method of claim 21 wherein the biological sample is selected fromthe group consisting of whole blood, white cells, mononuclear cells,platelet concentrates, blood, plasma, serum, cerebrospinal fluid, urine,saliva, milk, ductal fluid, tears, semen, feces, tonsils, lymph nodes,collagen, brain extracts and gland extracts.
 23. The method of claim 21wherein the ligand is attached to a solid support prior to contactingthe sample.
 24. The method of claim 23 wherein the solid support isselected from the group consisting of membranes and resins.
 25. Themethod of claim 23 wherein the solid support is a resin selected fromthe group consisting of polymethacrylate, agarose, sepharose,cross-linked agarose, composite cross-linked polysaccharides, celite,polyvinyl D, fluoride acrylate, polystyrene and cellulose.
 26. Themethod of claim 23 wherein the solid support is polymethacrylate resin.27. The method of claim 23 wherein the solid support is a membraneselected from the group consisting of nylon and cellulose.
 28. A methodof removing a prior protein from a sample, comprising: contacting thesample with a ligand capable of binding to one or more peptides orpolypeptides derived from a prion protein selected from the groupconsisting of PrPc, PrPsc and PrPr, under conditions sufficient to causeformation of a complex between the prion protein and the ligand; andremoving the complex from the sample.
 29. The method of claim 28 whereinthe sample is a biological sample.
 30. The method of claim 28 whereinthe biological sample is selected from the group consisting of wholeblood, white cells, mononuclear cells, platelet concentrates, blood,plasma, serum, cerebrospinal fluid, urine, saliva, milk, ductal fluid,tears, semen, feces, tonsils, lymph nodes, collagen, brain extracts andgland extracts.
 31. The method of claim 28 wherein the ligand isattached to a solid support prior to contacting the sample.
 32. Themethod of claim 28 wherein the solid support is selected from the groupconsisting of membranes and resins.
 33. The method of claim 28 whereinthe solid support is a resin selected from the group consisting ofpolymethacrylate, agarose, sepharose, cross-linked agarose, compositecross-linked polysaccharides, celite, polyvinyl D, fluoride acrylate,polystyrene and cellulose.
 34. The method of claim 28 wherein the solidsupport is polymethacrylate resin.
 35. The method of claim 28 whereinthe solid support is a membrane selected from the group consisting ofnylon and cellulose.
 36. A composition for binding prion proteins,comprising: a ligand capable of binding to one or more prion peptides;and a solid support, wherein the ligand is attached to the solidsupport.
 37. The composition of claim 36 wherein the solid support isselected from the group consisting of membranes and resins.